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Sep 2016

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Microarray, IPA and GSEA Analysis in Mice Models
关于小鼠模型的微阵列,IPA和GSEA分析    

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Abstract

This protocol details a method to analyze two tissue samples at the transcriptomic level using microarray analysis, ingenuity pathway analysis (IPA) and gene set enrichment analysis (GSEA). Methods such as these provide insight into the mechanisms underlying biological differences across two samples and thus can be applied to interrogate a variety of processes across different tissue samples, conditions, and the like. The full method detailed below can be applied to determine the effects of muscle-specific Notch1 activation in the mdx mouse model and to analyze previously published microarray data of human liposarcoma cell lines.

Keywords: Microarray (微阵列), Ingenuity pathway analysis (创新途径分析), Gene set enrichment analysis (基因集富集分析), Knowledge-based software (基于知识的软件), Transcriptomic-based analysis (转录组分析)

Background

Transcriptomic analysis of various cell types is crucial to elucidate the functional elements of a cell, provides insight into cell-specific characteristics and can highlight changes associated with different development or disease stages (Wang et al., 2009). While RNA-sequencing has become increasingly popular, the relative cost and time to analysis may be a burden. Therefore, microarray analysis is an alternative tool for comparing relative gene-expression levels between various mRNA samples (Read et al., 2001). Microarray is commonly used to investigate changes associated with disease states whose gene expression patterns can be inferred or have already been defined (Amaratunga et al., 2007). Ingenuity pathway analysis (IPA, QIAGEN) is commonly used in conjunction with large-scale omics data and provides information about pathways, genes and other signatures that may be significantly altered across different samples. Gene set enrichment analysis (GSEA) uses gene sets and characteristics that have been a priori associated with various diseases or pathways in order to provide biological application to the sample of interest.

The methods described below were used by Bi and colleagues to understand the effects of Notch signaling in muscle regeneration and liposarcoma, a common soft-tissue cancer type (Bi et al., 2016a). These methods probed the effects of myofiber-specific Notch activation in a Duchenne’s muscular dystrophy (mdx) mouse model and discovered that over-activation of Notch in the mdx mouse model displayed similar gene-expression patterns as healthy human muscle. Similarly, Bi and colleagues performed microarray analysis, IPA and GSEA to find that over-activation of Notch in mouse inguinal white adipose tissue shares signatures of human liposarcoma (Bi et al., 2016b). Both of these studies underscore the importance of comparative analyses when using animal models and since many microarray datasets are available online, gene set enrichment analysis (GSEA) can be used to evaluate already published datasets with respect to the investigator’s interest at relatively low cost. Discoveries such as these are imperative towards developing therapeutic targets and furthering our understanding of biological processes and how their perturbance may influence human disease.

Materials and Reagents

  1. RNA isolation and cDNA synthesis
    1. 1.5 ml microcentrifuge tubes (DOT Scientific, catalog number: RN1700-GMT )
    2. Mouse (strains purchased from the Jackson lab and used in this study: mdx (stock# 007914) and Adiponectin-Cre; transgenics were in a C57BL/6J and 129S4 mixed background)
    3. Liquid nitrogen
    4. Chloroform (Sigma-Aldrich, catalog number: 288306 )
    5. Isopropanol (Fisher Scientific, catalog number: S25372 )
    6. TRIzolTM reagent (Thermo Fisher Scientific, catalog number: 15596026 )
    7. 75% Ethanol (diluted in RNase-free water)
    8. Nuclease-free water (Thermo Fisher Scientific, catalog number: AM9916 )
    9. Optional: RNaseZapTM RNase Decontamination Solution (Thermo Fisher Scientific, catalog number: AM9780 ) or other RNase decontamination solution
    10. RNaseOUTTM Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific, catalog number: 10777019 )

  2. Real-Time Quantitative PCR
    1. LightCycler 480 96-well Multi-well Plate (Roche Diagnostics, catalog number: 04729692001 )
    2. LightCycler 480 Sealing Foil (Roche Diagnostics, catalog number: 04729757001 )
    3. 1.5 ml microcentrifuge tube (DOT Scientific, catalog number: RN1700-GMT )
    4. 10 mM dNTP set (Thermo Fisher Scientific, catalog number: 10297018
    5. RNaseOUTTM Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific, catalog number: 10777019 )
    6. Oligo(dT)18 primer (IDTDNA)
    7. M-MLV Reverse Transcriptase (Thermo Fisher Scientific, InvitrogenTM, catalog number: 28025013 )
      1. 5x First-Strand Buffer
      2. DTT
    8. SYBR Green Master Mix (Roche Diagnostics, catalog number: 4913854001 )
    9. Gene-specific primers and house-keeping gene-specific primers (i.e., 18S ribosomal subunit) ordered from IDTDNA.
      Note: Primers used to validate microarray results and for real-time quantitative PCR in Bi et al. (2016b) are listed in Supplemental file.

  3. Microarray
    1. Agilent SurePrint G3 Mouse GE 8 x 60 K chip (Agilent Technologies, catalog number: G4126A ; other chips can be used depending on tissue sample)
    2. Triton X-102 (Sigma-Aldrich, catalog number: X102-500mL )
    3. 100% isopropyl alcohol (Fisher Scientific, catalog number: S25372 )
    4. RNeasy Mini Kit (QIAGEN, catalog number: 74104 )
      1. 50 RNeasy Mini Spin Columns
      2. Collection tubes
      3. RNase-free Buffer RLT
        Add 10 μl β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 ) 1 ml of RPE buffer
      4. RNase-free Buffer RPE (concentrate)
        Add 4x volume of 100% Ethanol to buffer
      5. Buffer RW1
      6. RNase-free water
    5. 75% Ethanol (100% ethanol diluted in Nuclease-free water)
    6. Other microarray kit components 
      1. Cyanine 3-CTP and cyanine 5-CTP
      2. Spike A and Spike B Mix
      3. Dilution buffer
      4. T7 primer
      5. 5x First strand buffer
      6. 0.1 M DTT
      7. 10 mM dNTP mix
      8. Affinity Script RNase Block Mix
      9. NTP Mix
      10. T7 RNA Polymerase Blend
      11. Nuclease-free water
      12. 5x Transcription Buffer
      13. 10x blocking agent
        1,250 μl Nuclease-free water to 10x gene expression blocking agent 
      14. 1x HiRPM Hybridization buffer
        Equal volume 2x HiRPM Hybridization buffer to Nuclease-free water
      15. Gene expression Wash Buffers 1 and 2
      16. Triton X-102 (10%)
      17. RNeasy Mini Kit (see components listed above)
      18. Slide staining dishes
      19. Slide racks

Equipment

  1. RNA isolation and cDNA synthesis
    1. Balance 
    2. Pipettes (1,000 μl, 200 μl)
    3. Vortex (e.g., Scientific Industries, model: Vortex-Genie 2 , catalog number: SI0236)
    4. Centrifuge capable of reaching 16,000 x g and 4 °C for 1.5 ml tubes (Eppendorf, model: 5424 R )
    5. Tissue homogenizer (e.g., Fisher Scientific, catalog number: FB120110 )
    6. Heat block capable of maintaining following temperatures for 1.7 ml micro-centrifuge tubes: 65 °C, 37 °C, 25 °C, 70 °C
    7. Water bath (e.g., Thermo Fisher Scientific, catalog number: TSGP02 )
    8. Optional: Rotating incubator 
    9. Optional: Rotator rack

  2. Microarray
    1. Stir bar
    2. Pipettes (1,000 μl, 200 μl, 10 μl, multi-channel pipette)
    3. Centrifuge (Eppendorf, model: 5424)
    4. Agilent Technology Surescan Microarray Scanner
    5. Hybridization oven (Agilent Technologies, catalog number: G2545A )
    6. NanoDropTM ND-1000 UV-VIS Spectrophotometer version 3.2.1 or higher (Thermo Fisher Scientific, model: NanoDropTM 1000 , catalog number: ND-1000)
    7. Heat block capable to maintaining following temperatures for 1.7 ml micro-centrifuge tubes: 80 °C, 70 °C, 60 °C , 40 °C, 37 °C
    8. Agilent Gene Expression Two Color Microarray (Agilent Technologies, catalog number: G4140-90050 )

  3. RNA quantification and quality control
    1. NanodropTM 2000c (or spectrophotometer) (Thermo Fisher Scientific, model: NanoDropTM 2000c )
    2. Agilent Bioanalyzer 2100

  4. qRT-PCR
    1. Roche Light Cycler 480 PCR System for 96-well plate

Software

  1. SAS software for statistical analysis (https://www.sas.com/en_us/software/university-edition.html)
  2. OligoAnalyzer 3.1 (IDT) (https://www.idtdna.com/calc/analyzer)
  3. GSEA (Gene set enrichment analysis) software available at: http://software.broadinstitute.org/gsea/index.jsp
  4. IPA (Ingenuity pathway analysis) software available at: https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/
  5. Agilent Microarray Scan Control (provided with current instrumentation)
  6. Agilent Feature Extraction Software 12.0 (provided with current instrumentation)
  7. Agilent GeneSpring GX software (provided with current instrumentation)
  8. NCBI Blastn (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch)
  9. UCSC Genome Browser, In silico PCR tool (https://genome.ucsc.edu/)

Procedure

  1. RNA isolation
    Notes: 
    1. Isolate no less than 500 ng RNA from Mus musculus adult whole tibialis anterior (TA) skeletal muscle (Figure 1).


      Figure 1. Image of tibialis anterior (TA) anatomy and dissection. Dashed red lines represent location of the TA. A. Location of TA prior to the removal of fur; B. Location of TA prior to dissection of the muscle; C. TA is cut from the tendon near the foot and pulled up towards the knee. D. Image of fully dissected TA.

    2. Maintenance of RNase-free conditions are imperative to high quantity and quality yield of RNA from tissue, and surfaces can be cleaned with RNaseZapTM. RNA will be used for microarray analysis and cDNA synthesis.
    3. Expect yield > 4 μg RNA/sample
    1. Dissect TA muscle or tissue of interest from respective mouse specimen (Figures 1A-1D).
    2. Weigh TA muscle, flash freeze in liquid nitrogen and add 1 ml of TRIzolTM reagent per 50-100 mg of tissue in 1.5 ml micro-centrifuge tube.
    3. Use a tissue homogenizer to homogenize the tissue for 2-3 sec at a time, placing the sample on ice to ensure it does not over-heat. The sample should be homogenized until no clear pieces of tissue are visible, approximately 2-4 times.
    4. Optional: clear lysate by centrifugation for 5 min at 4 °C and 12,000 x g and transfer supernatant to a new 1.5 ml microcentrifuge tube (approximate volume ~800 μl).
    5. Incubate the sample at room temperature for 5 min.
    6. Add 0.2 ml of chloroform per 1 ml of TRIzolTM reagent used, and shake or vortex vigorously for 15 sec. Then incubate the sample at room temperature for 3 min.
    7. Centrifuge for 15 min at 4 °C and 12,000 x g to separate phases.
    8. Transfer the aqueous phase (clear, upper phase Figure 2) containing the RNA to a new tube using a 1 ml pipette (volume ~400-600 μl), making sure not to transfer any of the interphase or phenol-chloroform phase.


      Figure 2. Image of phase separation following TRIzolTM reagent RNA extraction. Aqueous phase (gray) contains RNA and is to be carefully transferred into a fresh tube for downstream isolation.

    9. Add 0.5 ml of isopropanol per 1 ml of TRIzolTM reagent used to the aqueous phase containing RNA from Step A8 and incubate at room temperature for 10 min.
    10. Centrifuge sample for 10 min at 4 °C and 12,000 x g to precipitate RNA (RNA should form a white pellet at the bottom of the tube).
    11. Discard the supernatant being careful not to disturb the pellet.
    12. Resuspend pellet in 1 ml of 75% ethanol per 1 ml of TRIzolTM reagent used to wash the RNA pellet.
    13. Gently shake sample briefly and centrifuge for 5 min at 4 °C and 7,500 x g.
    14. Carefully discard supernatant, removing as much ethanol as possible without disturbing the pellet.
    15. Air-dry pellet for maximum 5-10 min at room temperature. Make sure that the ethanol has evaporated but do not let the pellet dry for too long as residual ethanol and over-drying both may affect RNA quality. 
    16. Resuspend pellet in 50 μl of RNase-free water (imperative if being used for downstream microarray analysis).
    17. Measure concentration and the ratio of A260/A280 using NanoDrop Spectrophotometer.
      Notes:
      1. One milligram of the sample should yield roughly 1 μg of RNA.
      2. The ratio of A260/A280 in the range of 1.85-1.95 is required for downstream applications, and the RNA sample quality should be checked using the Agilent bioanalyzer.
      3. Determination of the quality of RNA sample on an Agilent bioanalyzer should yield two large peaks corresponding to the 18S and 28S ribosomal subunits (Peterson et al., 2009). Examples of good and poor quality RNA are shown in Figure 3.


        Figure 3. Images of intact (ideal), partially degraded and degraded RNA samples as determined by using the Agilent BioAnalyzer. The top panel clearly shows two peaks which correspond to the 18S and 28S ribosomal subunits, a small peak to a spike-in control and otherwise smooth lines. The middle and bottom panels represent degraded RNA in which the 18S and 28S peaks may or may not be clear and are usually preceded by multiple smaller peaks (indicative of degraded RNA). The x- and y-axis are nucleotides and fluorescence, respectively.

  2. Transcriptomic Microarray Analysis
    Microarray methods and analysis are adapted from the manufacturer’s manuals (Agilent, Two-Color Microarray-Based Gene Expression Analysis–Low Input Quick Amp Labeling) 
    1. Preparation of Spike A Mix and Spike B Mix as positive controls, all procedures should be done in an RNase-free environment to ensure stability of RNA, following steps can be done in a 1.5 ml microcentrifuge tube.
      1. Vortex and heat Spike A and B Mix at 37 °C for 5 min upon arrival and briefly centrifuge.
      2. To dilute Spike A Mix (for example) prepare first dilution by adding 2 μl of Spike A Mix to 38 μl dilution buffer, mix, and briefly spin down (dilution is 1:20).
      3. Dilute solution from Step B1 b by adding 2 μl of diluted solution to 78 μl dilution buffer, mix, and briefly spin down (dilution is 1:40).
      4. Dilute solution from Step B1 c by adding 2 μl of diluted solution to 30 μl dilution buffer, mix, and briefly spin down (dilution is 1:16).
      5. Dilute solution from Step B1 d by adding 4 μl of diluted solution to 28 μl dilution buffer, mix, and briefly spin down (dilution is 1:8).
      6. Add 2 μl of final diluted solution (final dilution is 1:102,400) to 25-100 ng of sample RNA (volume should not exceed 3.5 μl).
      7. Repeat for Spike B Mix for other RNA samples (i.e., RNA from wild-type sample vs. RNA from experimental sample) and proceed with labeling reaction.
    2. Labeling reaction, purification and quantification of fluorescently labeled complementary RNA (cRNA)
      Note: For Spike A prepare with Cyanine 3-CTP and Spike B Cyanine 5-CTP dye otherwise both samples are treated the same. Have water baths or heating blocks set to 65 °C and 80 °C prior to starting procedure for Steps B2a-B2d (below). 
      1. Prepare T7 primer mix by combining 1.8 μl T7 primer to 1 μl nuclease-free water per reaction.
      2. Add 1.8 μl T7 primer mix to each tube and incubate at 65 °C for 10 min, gently shaking the tube every couple of minutes.
      3. Remove the reactions from heat and place on ice for 5 min.
      4. In the meantime, pre-warm the 5x first strand buffer at 80 °C for 3-4 min, vortex and spin down so that the buffer components are fully re-suspended.
      5. Assemble the following cDNA reaction on ice in a 1.5 ml microcentrifuge tube (scaling up according to the number of reactions with one reaction in excess to correct for pipetting error):

      6. Add 4.7 μl cDNA reaction to each tube containing RNA + Spike Mix (final volume is 10 μl), mix by pipetting and briefly spin down.
      7. Pre-heat blocks to 42 °C and 70 °C 10 min prior to use to ensure temperature reaches desired degrees (i.e., 42 °C and 70 °C). 
      8. Incubate reaction at 40 °C for 2 h then heat inactivate reaction at 70 °C for 15 min (occasionally shaking tube or using a circulating water bath).
      9. Place the sample on ice for 5 min and in the meantime prepare the transcription master mix reactions (scaling up according to the number of reactions) as follows to amplify and fluorescently label the RNA:

      10. Add 6 μl of transcription reaction to Spike A Mix or Spike B Mix (final volume per reaction now 16 μl), mix by pipetting up and down and incubate at 40 °C for 2 h.
      11. Using the RNeasy Mini Kit, purify the cRNA from each reaction (protocol below as summarized per the manufacturer’s instructions)
        1. Bring volume of cRNA reaction to 100 μl with nuclease-free water.
        2. Add 350 μl of RLT and mix well.
        3. Add 250 μl of 100% ice cold ethanol and mix by gently pipetting.
        4. Transfer reaction to spin column, place in a collection tube and centrifuge at 16,000 x g and 4 °C for 30 sec.
        5. Discard flow through and add 500 μl of RPE buffer to the column, centrifuge at 16,000 x g and 4 °C for 30 sec.
        6. Repeat Steps B2j-B2k v but centrifuge at 16,000 x g and 4 °C for 60 sec.
        7. Place column in a fresh collection tube and centrifuge at 16,000 x g and 4 °C for 30 sec to remove and remaining buffer.
        8. Place column in a 1.5 ml microcentrifuge tube and add 30 μl of RNase-free water to the column.
        9. Incubate at room temperature for 1-2 min and then centrifuge at 16,000 x g and 4 °C for 30 sec.
        10. Optional: re-elute with eluate to increase yield.
        11. Measure RNA concentration and quality with a NanoDropTM ND-1000 UV-VIS Spectrophotometer version 3.2.1 or higher.
          1)
          Use 'Microarray Measurement' tab and select RNA-40 as sample type.
          2)
          Record cyanine 3 or 5 concentration, RNA absorbance ratio and cRNA concentration (RNA absorbance ratio [260/280 nm] should be 1.9 ± 0.04 and cRNA concentration should yield at minimum 1.875 μg if using 2-pack format).
    3. Hybridization of sample and probes
      1. Preparation of 10x blocking: add 1,250 μl of nuclease-free water to the 10x gene expression blocking agent (supplied with the kit) and gently vortex to dissolve powder completely.
        Note: If powder does not readily dissolve, heat blocking agent at 37 °C for 4-5 min.
      2. Assemble the following reaction in a 1.5 ml microcentrifuge tube.
        Note: The reaction below and subsequent volumes are for 2-pack microarray, for 1-pack, 4-pack or 8-pack refer to refer to Agilent’s Two-color Microarray-Based Gene Expression Analysis Protocol.

      3. Incubate at 60 °C for 30 min then place reaction on ice.
      4. Add equal volume 2x Hi-RPM Hybridization Buffer to stop reaction and mix well, being careful to not introduce any bubbles.
      5. Briefly spin sample down and place on ice for immediate downstream use.
      6. Load gasket slide into Agilent SureHyb (Figures 4B and 4C) chamber base with label facing up.
      7. Slowly add 240 μl of sample onto the gasket well from left to right, being careful not to introduce any air bubbles.
      8. Make 1x solution from 2x Hi-RPM Hybridization Buffer in any wells that remain unused.
      9. Place the slide with the ‘active’ side down, such that the Agilent-labeled barcode is facing down and the numeric barcode facing upwards.
      10. Place the SureHyb chamber cover onto the slides, clamp both pieces and tighten.
      11. Double-check that there are no stationary bubbles and, if needed, tap on surface to remove.
        Note: bubbles may be a source of artifacts as they may impact signal intensity.
      12. Load the chamber onto the rotator rack in the hybridization oven, set it to rotate at 10 rpm and hybridize at 65 °C for 17 h.
    4. Washing microarray slides
      1. To prepare the Wash Buffers, remove outer and inner caps from container and use a pipette to add 2 ml of Triton X-102 to gene expression Wash Buffers 1 and 2.
      2. Mix by inversion and replace the original outer and inner caps with the spigot provided with the kit.
      3. Pre-warm gene expression Wash Buffer 2 to 37 °C before proceeding with washing the arrays.
      4. Wash the staining dish prior to use as follows (repeat 2 x):
        1. Add slide rack and stir bar to staining dish and fill the dish with 100% isopropyl alcohol (Figure 4A).
        2. Turn on magnetic stir plate to wash for 5 min.
        3. Rinse staining dish with Milli-Q water multiple times.


          Figure 4. Image of microarray wash dishes, SureHyb chamber and slide. A. Image of the wash dish with metal stir bar (left) and slide holder. B. Assembled SureHyb chamber, cover and clamp. C. Individual parts of the SureHyb assembly kit. Top is the chamber, the microarray slide sits on top of it and the cover (middle) is gently placed on top. The clamp (bottom) is then used to ensure the slide and chamber stay tightly together. D. Image of microarray slides, the visible barcodes clearly state “Agilent” and are to be used for proper orientation.

      5. Prepare staining dishes as follows
        1. Fill slide-staining dish #1 with gene expression Wash Buffer 1.
        2. Place slide rack into slide-staining dish #2 and add magnetic stir bar, fill with gene expression Wash Buffer 1 and place on a magnetic plate.
        3. Place dish #3 on the stir plate, add a stir bar and only fill with pre-warmed gene expression Wash Buffer 2 immediately before use.
      6. Remove hybridization chamber from the rotating incubator and note any bubbles that may have formed during hybridization.
      7. Disassemble the hybridization chamber by placing it on a flat surface, remove the array-gasket while maintaining the numeric barcode facing up and immediately submerge it in slide-staining dish #1.
      8. Keeping the array-gasket sandwich submerged, pry open the sandwich with forceps and let the gasket slide drop to the bottom of the dish.
      9. Remove the slide and place it into the slide rack in slide-staining dish #2, being careful to only touch the slide over the numeric barcode or along the thin edges.
      10. Repeat these steps for the remaining slides.
      11. Incubate the slide on the magnetic stir plate for 1 min.
      12. Add slide rack to slide-staining dish #3 and incubate on the magnetic stir plate for 1 min.
      13. Slowly and carefully remove slide rack and place slides on the slide holder.
        1. Add the slide without the barcode label towards the edge.
        2. Active microarray surface should be facing up towards the slide cover.
        3. Close the plastic cover.
      14. Proceed to scanning slides.
    5. Scanning microarray slides, feature selection and data collection
      1. Place the slide holder containing slide into the scanner cassette.
      2. Select the ‘AgilentG3_HiSen_GX_2color’ protocol.
      3. Click ‘Start scan’.
      4. Open Agilent Feature Extraction (FE) and add the images to be extracted to the FE project (default settings for project ok).
        Note: Manual grid mapping may be required.
      5. Save the Feature Extraction project as .fep via File > Save As.
      6. Select Project > Start Extracting.

  3. Validation of microarray results
    1. Gene-specific primer design for real-time quantitative PCR (qPCR): selection of amplicon size, primers and template are imperative to generating reproducible data that can accurately determine if the results from the microarray are validated
      Note: Pre-validated gene expression assays can be purchased from a variety of vendors and thus do not require optimization. For genes that are not available or have not been previously validated, primers efficiency should be evaluated (see below):
      1. Select genes based on the microarray results. Candidate genes should be chosen based on genes that displayed a significant change across conditions and a reference gene should be chosen that did not display any change in expression across samples (for the latter examples include 18S rRNA or β-actin).
      2. Amplicons targeted by primer should be approximately equal in size (not greater than 0.6 kb) and the secondary structures of the target sites can be determined using nucleic acid-folding software such as OligoAnalyzer 3.1 (IDT), as highly structured sequences can impact qPCR efficiency and results.
      3. Primers target sites should be analyzed by in silico PCR tools such as NCBI BLAST or UCSC Genome Browser to determine specificity.
      4. Each primer should have roughly the same melting temperature, however the exact ideal annealing temperature must be determined experimentally.
    2. cDNA synthesis
      1. One to five microgram of template RNA from tissue samples assayed in microarray analysis required (use both biological and technical replicates here; the former being another mouse sample under the same condition and the latter the same RNA that was used for microarray analysis), and quality should be determined prior to cDNA synthesis.
      2. Thaw reagents from M-MLV RT kit, vortex and centrifuge briefly.
      3. Assemble the following reaction on ice in the respective order:

      4. Gently flick PCR tubes to mix contents, briefly centrifuge and incubate at 65 °C for 5 min then place on ice immediately.
      5. Add the following components to the reaction in the respective order:

      6. Mix reaction by pipetting up and down and incubate at 25 °C for 10 min.
      7. Incubate reaction at 37 °C for 50 min.
      8. Heat inactivate reaction at 70 °C for 15 min.
      9. Resulting cDNA can be used immediately for real-time PCR analysis or stored at -20 °C.
    3. Real-time qPCR of candidate target genes
      Note: Important to set up biological and technical replicates as well as a negative control containing no template. 3 biological replicates (i.e., three RNA/cDNA samples from three different mice), 3 technical replicates (i.e., using the same RNA to generate cDNA) suggested per sample.
      1. Primers should be re-hydrated in nuclease-free water and stored at a stock concentration of 10 μM.
      2. Thaw reagents on ice and prepare the following reaction on ice (multiply the final reaction volume by the number of PCR reactions planned plus to [to account for pipetting error] create a master mix that can be aliquoted into Roche LightCycler 480 plates).

      3. General Real-time Quantitative PCR cycler settings.

      4. Cq values can then be analyzed for primer efficiency and subsequent fold-change in target gene expression (Cq values > 35 are not recommended to use).
        Note: Samples requiring over 35 cycles may not provide reliable results and indicate that the cDNA quality or reaction efficiency is poor. While some very lowly expressed genes may yield a Cq value between 35-40, under those conditions it is imperative the negative control produces no signal at that cycle number

Data analysis

This part of the protocol includes the analysis of the microarray results and subsequent gene-set enrichment and ingenuity pathway analyses to determine candidate genes and enriched biological pathways/processes (respectively). Following candidate gene selection, real-time PCR analysis is performed to validate candidates (for an overview of workflow see Figure 5).


Figure 5. Overview of the workflow for microarray analysis. RNA is isolated from tissue samples (or cells) of interest. Quality of RNA is determined prior to proceeding with generation of cRNA, hybridization and data acquisition. Analysis of the microarray data is performed by the Agilent software. From there, the Gene Set Enrichment Analysis software is employed to yield genes that are significantly enriched in the assayed sample. Microarray data will also be used for Ingenuity Pathway Analysis (IPA) (and can be used for Gene Ontology [GO] analysis) to reveal pathways or biological processes that are enriched in the target sample.


  1. Analysis of microarray results
    Normalization, gene alignments and calls (to correlate gene expression levels) and evaluation of genes with statistically significant gene expression changes across the evaluated samples.
    1. Download GeneSpring software to perform statistical analysis and open software.
    2. Create a new project, load the text files from the feature extractor (FE) and click ‘Next’, keeping the software settings as default.
    3. The data will be uploaded onto GeneSpring upon clicking ‘Finish’.
    4. Assign ‘Experimental Grouping’ and then ‘Create an Interpretation’ with the respective experimental groupings properly selected.
    5. Statistical analysis
      1. Perform an Analysis of variance (ANOVA) using the software, selecting a Tukey post-hoc test and the appropriate pairing options (depending on samples).
      2. If comparing wild-type and a knock out-sample, perform a Student’s t-test (in general, the statistical test performed will depend on the samples).
    6. Fold-change analysis: elimination of probes that do not meet 1.5 fold-change.
      1. Select ‘Fold-change’ with the same interpretation as used for the ANOVA.
      2. Adjust fold-change to 1.5 and to determine how many probes meet this criterion.
      3. Once ‘Finish’ has been selected, the probe lists should appear.
    7. Combine the probes whose expression increases or decreases into one excel sheet for ingenuity pathway analysis.

  2. Ingenuity Pathway Analysis (IPA)
    IPA is used to determine pathways that may be altered across samples based on microarray results.
    1. Login to IPA via www.ingenuity.com/products/ipa.
    2. Upload excel file that was saved from GeneSpring to software, select ‘Agilent’ and the appropriate identified type (i.e., appropriate species).
    3. Probe sample should be ‘ID’ and logFoldChange should be ‘Observation 1’.
    4. Click ‘Continue’, and then use the ‘Analysis-ready’ list to select ‘Run Analysis’.
    5. Gene ontology information will then appear and can be analyzed for pathway enrichment across samples.

  3. Gene Set Enrichment Analysis (GSEA)
    GSEA is a computational evaluation of whether the gene expression differences across biological samples among certain gene sets reach statistical significance. This requires input of the microarray data results and selection of reference dataset for GSEA (Subramanian et al., 2005).
    1. Results from the microarray analysis should be filtered to yield a list of genes that display a ≥ 1.5-fold change in expression across experimental samples and reach a significance level with the corrected P-value of ≤ 0.05.
    2. Download GSEA software and install per the manual’s instructions (available for both R and Java).
    3. Determine the reference gene set on which the analysis should be performed: e.g., for Bi et al. (2016a) the human DMD gene expression dataset was compared to healthy human muscle which was chosen in order to understand the applicability of results to mouse models (NCBI dataset GDS3027).
    4. Microarray results containing expression data from Step C1 should be converted to the GCT file-type: Details on how to convert files to the required type for GSEA can be found on GenePattern: file format guide.
    5. A gene set database file containing a reference dataset to analyze against and a sample phenotype file must also be generated for input into GSEA (as well as an empty directory to store the output results)
      1. Gene set database file (.gmt) formatting details can be found here on GenePattern: file format guide.
      2. Sample phenotype file (.cls) formatting details can be found here on GenePattern: file format guide.
    6. For the analysis used in Bi et al. (2016a and 2016b), default settings were used when calling GSEA.
      Note: However, these can be changed as seen fit; see source code documentation for more details.
    7. GSEA results and graphical analysis
      1. The output directory should contain GSEA summary results file; determine that the parameters meet the specified values prior to proceeding (ideal values can be found at Subramanian et al., 2005).
      2. GSEA R package contains GSEA.Analyze.Sets which generates plots of the input data, refer to the source code documentation for specific parameters.

  4. Analysis of real-time PCR results (Bustin et al., 2009) (used for validation of target genes as discovered by microarray and GSEA)
    1. Primer efficiency
      1. All primers used for real-time PCR should be assessed for efficiency.
      2. All primers should amplify a target site with similar efficiency with respective to the reference. Efficiency of the primer amplification can be determined by generating a calibration curve.
        1. Briefly, calibration curves can be determined by reverse-transcription of high-quality RNA into cDNA. cDNA is then serially diluted by a factor of 10 4-5 times (i.e., 1:10, 1:100, 1:1,000, 1:10,000) and the primers are assayed on each dilution. 
        2. Plot initial log cDNA concentration vs. Cq value

          Cq = m(log cDNA concentration) + b

          where, m = slope, b = y-intercept
        3. Determine PCR efficiency
          Efficiency is measured as 10-1/slope-1 
    2. Analysis of results
      1. The ∆∆Cq is used to determine the change in expression compared to the reference gene.
        1. Normalize the Cq value of candidate gene to reference gene (∆Cq).
        2. Transform normalized ∆Cq exponentially (log2(∆Cq)).
        3. Take the average of the technical replicates (i.e., three individual reactions from same biological sample) and determine standard deviation; guidelines provided in Bustin et al. (2009).
        4. Normalize averaged results and standard deviations to reference gene.
        5. Fold-change is then (1-∆∆Cq) x 100.
      2. Fold-change directionality should be consistent with results from microarray analysis.

Notes

  1. Validation of microarray results using real-time quantitative PCR
    1. Validation of microarray results using real-time quantitative PCR against target genes should be done prior to GSEA since it is essential to ensure that the results obtained from the microarray can be reproduced via an alternate approach (thus substantiating their biological significance). 
    2. All thresholds for gene expression and real-time quantitative PCR are detailed in the above protocol. Likewise, it is imperative to have positive and negative controls, as well as biological and technical replicates in order to reach statistical significance (generally, at least three replicates/sample, however power analysis should be used to determine the sample size). For example, a negative control reaction when generating cDNA should be used for subsequent real-time quantitative PCR to confirm no contaminating materials. 
    3. Microarray results can be validated by real-time quantitative PCR using the same RNA used for microarray analysis. However, a power analysis should be conducted to determine the appropriate sample size for each experiment and a Student’s t-test with a two-tail distribution can be to analyze results unless specified otherwise. 
  2. Validation of microarray results using Gene-ontology (GO) term analysis
    Gene-ontology (GO) term analysis can also be performed on the list of genes generated from the microarray; however GSEA provides a rank and weight to each gene such that relative expression level in the sample is taken into consideration thus helping researchers identify candidate genes. GO term analysis does not provide gene-specific information however both GSEA and GO-term analysis will yield biological pathways that are significantly enriched in the assayed samples

Acknowledgments

The study is partially supported by NIH grants R01CA212609 and R01AR071649. Protocol was adapted from Bi et al. (2016a and 2016b) listed in the References below.

Competing interests

The authors declare no conflicts of interest or competing interests.

Ethics

All procedures involving the use of animals were performed in accordance with the guidelines presented by Purdue University’s Animal Care and Use Committee (PACUC).

References

  1. Amaratunga, D., Göhlmann, H. and Peeters, P. J. (2007). 3.05 – Microarrays. In: Taylor, J. B. and Triggle, D. J. (Eds.). Comprehensive Medicinal Chemistry II. Elsevier, 87-106.
  2. Bi, P., Yue, F., Karki, A., Castro, B., Wirbisky, S. E., Wang, C., Durkes, A., Elzey, B. D., Andrisani, O. M., Bidwell, C. A., Freeman, J. L., Konieczny, S. F. and Kuang, S. (2016b). Notch activation drives adipocyte dedifferentiation and tumorigenic transformation in mice. J Exp Med 213(10): 2019-2037.
  3. Bi, P., Yue, F., Sato, Y., Wirbisky, S., Liu, W., Shan, T., Wen, Y., Zhou, D., Freeman, J. and Kuang, S. (2016a). Stage-specific effects of Notch activation during skeletal myogenesis. Elife 5: e17355.
  4. Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M. W., Shipley, G. L., Vandesompele, J. and Wittwer, C. T. (2009). The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55(4): 611-622.
  5. Peterson, S. M. and Freeman, J. L. (2009). RNA isolation from embryonic zebrafish and cDNA synthesis for gene expression analysis. J Vis Exp (30): 1470.
  6. Read, J. and Brenner, S. (2001). Microarray Techonlogy. In: Brenner, S. and Miller, J. H. (Eds.). Encyclopedia of Genetics. Academic Press 1191.
  7. Wang, Z., Gerstein, M. and Snyder, M. (2009). RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10(1): 57-63.

简介

该协议详述了使用微阵列分析,独创性途径分析(IPA)和基因集富集分析(GSEA)在转录组水平分析两个组织样品的方法。 诸如此类的方法提供了对两个样品之间的生物学差异的潜在机制的洞察,因此可以应用于跨越不同组织样品,条件等询问各种过程。 下面详述的完整方法可用于确定肌肉特异性Notch1激活在 mdx 小鼠模型中的作用,并分析先前公布的人脂肪肉瘤细胞系的微阵列数据。

【背景】各种细胞类型的转录组学分析对于阐明细胞的功能元件至关重要,可以深入了解细胞特异性特征,并可突出与不同发育或疾病阶段相关的变化(Wang et al。,2009) 。虽然RNA测序变得越来越流行,但分析的相对成本和时间可能是一种负担。因此,微阵列分析是比较各种mRNA样品之间相对基因表达水平的替代工具(Read et al。,2001)。微阵列通常用于研究与疾病状态相关的变化,其疾病状态的基因表达模式可以被推断或已经被定义(Amaratunga et al。,2007)。 Ingenuity途径分析(IPA,QIAGEN)通常与大规模组学数据结合使用,并提供有关不同样本可能显着改变的途径,基因和其他特征的信息。基因集富集分析(GSEA)使用与各种疾病或途径相关的先验的基因集和特征,以便为感兴趣的样品提供生物学应用。

Bi及其同事使用下述方法来理解Notch信号传导在肌肉再生和脂肪肉瘤中的作用,脂肪肉瘤是一种常见的软组织癌类型(Bi et al。,2016a)。这些方法进行探测特定肌纤维刻活化在杜兴氏肌营养不良症的影响( MDX )小鼠模型,发现过度激活在 MDX 小鼠模型显示类似的Notch基因表达模式作为健康的人体肌肉。类似地,Bi和同事进行微阵列分析,IPA和GSEA地发现,过度激活在人类脂肪肉瘤的小鼠腹股沟白色脂肪组织股签名的Notch的(Bi 等人,2016B)。这两项研究都强调了使用动物模型时比较分析的重要性,并且由于许多微阵列数据集可在线获得,因此基因集富集分析(GSEA)可用于以相对较低的成本评估已发布的数据集,以及研究者的兴趣。诸如此类的发现对于制定治疗目标和进一步了解生物过程以及它们的干扰如何影响人类疾病是必不可少的。

关键字:微阵列, 创新途径分析, 基因集富集分析, 基于知识的软件, 转录组分析

材料和试剂

  1. RNA分离和cDNA合成
    1. 1.5 ml微量离心管(DOT Scientific,目录号:RN1700-GMT)
    2. 小鼠(从杰克逊实验室购买并在本研究中使用的菌株: mdx (股票#007914)和脂联素 -Cre;转基因在C57BL / 6J和129S4混合背景中)
    3. 液氮
    4. 氯仿(Sigma-Aldrich,目录号:288306)
    5. 异丙醇(Fisher Scientific,目录号:S25372)
    6. TRIzol TM 试剂(Thermo Fisher Scientific,目录号:15596026)
    7. 75%乙醇(在不含RNase的水中稀释)
    8. 无核酸酶水(Thermo Fisher Scientific,目录号:AM9916)
    9. 可选:RNase Zap TM RNase去污溶液(Thermo Fisher Scientific,目录号:AM9780)或其他RNase去污解决方案
    10. RNaseOUT TM 重组核糖核酸酶抑制剂(赛默飞世尔科技,目录号:10777019)

  2. 实时定量PCR
    1. LightCycler 480 96孔多孔板(罗氏诊断,目录号:04729692001)
    2. LightCycler 480密封箔(罗氏诊断,产品目录号:04729757001)
    3. 1.5 ml微量离心管(DOT Scientific,目录号:RN1700-GMT)
    4. 10mM dNTP组(Thermo Fisher Scientific,目录号:10297018) 
    5. RNaseOUT TM 重组核糖核酸酶抑制剂(Thermo Fisher Scientific,目录号:10777019)
    6. Oligo(dT) 18 引物(IDTDNA)
    7. M-MLV逆转录酶(Thermo Fisher Scientific,Invitrogen TM ,目录号:28025013)
      1. 5x First-Strand Buffer
      2. DTT
    8. SYBR Green Master Mix(罗氏诊断,目录号:4913854001)
    9. 从IDTDNA订购的基因特异性引物和管家基因特异性引物(即,18S核糖体亚基)。
      注:用于验证微阵列结果和Bi等人的实时定量PCR的引物。 (2016b)列在补充文件中。

  3. 微阵列
    1. Agilent SurePrint G3鼠标GE 8 x 60 K芯片(Agilent Technologies,目录号:G4126A;可根据组织样本使用其他芯片)
    2. Triton X-102(Sigma-Aldrich,目录号:X102-500mL)
    3. 100%异丙醇(Fisher Scientific,目录号:S25372)
    4. RNeasy Mini Kit(QIAGEN,目录号:74104)
      1. 50个RNeasy迷你旋转柱
      2. 收集管
      3. 无RNase缓冲液RLT
        加入10μlβ-巯基乙醇(Sigma-Aldrich,目录号:M6250)1 ml RPE缓冲液
      4. RNase-free Buffer RPE(浓缩液)
        加入4倍体积的100%乙醇缓冲液
      5. 缓冲区RW1
      6. 不含RNase的水
    5. 75%乙醇(在不含核酸酶的水中稀释100%乙醇)
    6. 其他微阵列试剂盒组件 
      1. 花青素3-CTP和花青5-CTP
      2. Spike A和Spike B Mix
      3. 稀释缓冲液
      4. T7底漆
      5. 5x第一链缓冲液
      6. 0.1 M DTT
      7. 10mM dNTP混合物
      8. 亲和脚本RNase Block Mix
      9. NTP Mix
      10. T7 RNA聚合酶混合物
      11. 不含核酸酶的水
      12. 5x转录缓冲液
      13. 10x阻断剂
        1,250μl无核酸酶水至10x基因表达阻断剂 
      14. 1x HiRPM杂交缓冲液
        等体积2x HiRPM杂交缓冲液至无核酸酶水
      15. 基因表达洗涤缓冲液1和2
      16. Triton X-102(10%)
      17. RNeasy Mini Kit(参见上面列出的组件)
      18. 滑动染色皿
      19. 滑动架子

设备

  1. RNA分离和cDNA合成
    1. 平衡 
    2. 移液器(1,000μl,200μl)
    3. Vortex(例如,Scientific Industries,型号:Vortex-Genie 2,目录号:SI0236)
    4. 离心机能够达到16,000 x g 和4°C用于1.5 ml管(Eppendorf,型号:5424 R)
    5. 组织匀浆器(例如,Fisher Scientific,目录号:FB120110)
    6. 能够保持1.7 ml微量离心管温度的加热块:65°C,37°C,25°C,70°C
    7. 水浴(例如,Thermo Fisher Scientific,目录号:TSGP02)
    8. 可选:旋转培养箱 
    9. 可选:旋转架

  2. 微阵列
    1. 搅拌棒
    2. 移液器(1,000μl,200μl,10μl,多通道移液器)
    3. 离心机(Eppendorf,型号:5424)
    4. 安捷伦科技Surescan微阵列扫描仪
    5. 杂交烤箱(Agilent Technologies,目录号:G2545A)
    6. NanoDrop TM ND-1000 UV-VIS分光光度计3.2.1或更高版本(Thermo Fisher Scientific,型号:NanoDrop TM 1000,目录号:ND-1000)
    7. 能够为1.7 ml微量离心管保持以下温度的加热块:80°C,70°C,60°C,40°C,37°C
    8. 安捷伦基因表达双色微阵列(Agilent Technologies,目录号:G4140-90050)

  3. RNA定量和质量控制
    1. Nanodrop TM 2000c(或分光光度计)(Thermo Fisher Scientific,型号:NanoDrop TM 2000c)
    2. Agilent Bioanalyzer 2100

  4. qRT-PCR
    1. 罗氏Light Cycler 480 PCR系统用于96孔板

软件

  1. 用于统计分析的SAS软件( https://www.sas.com/en_us/software /university-edition.html )
  2. OligoAnalyzer 3.1(IDT)( https://www.idtdna.com/calc/analyzer )
  3. GSEA(基因集富集分析)软件位于: http://software.broadinstitute.org/gsea/ index.jsp的
  4. IPA(Ingenuity途径分析)软件可从以下网址获得: https://www.qiagenbioinformatics.com/产品/独创性途径分析/
  5. 安捷伦微阵列扫描控制(随当前仪器提供)
  6. 安捷伦特征提取软件12.0(随当前仪器提供)
  7. 安捷伦GeneSpring GX软件(随当前仪器提供)
  8. NCBI Blastn( https://blast.ncbi.nlm.nih.gov/ Blast.cgi?PAGE_TYPE = BlastSearch )
  9. UCSC基因组浏览器, In silico PCR工具( https://genome.ucsc.edu/

程序

  1. RNA分离
    注意: 
    1. 从Mus musculus成人整个胫骨前肌(TA)骨骼肌中分离出不少于500 ng的RNA(图1)。


      图1.胫骨前肌(TA)解剖和解剖图像。虚线红线代表TA的位置。 A.移除毛皮之前TA的位置; B.解剖肌肉之前TA的位置; C. TA从脚附近的肌腱切开并向膝盖拉起。 D.完全解剖TA的形象。

    2. 维持无RNase条件对于从组织中获得高质量和高质量的RNA是必不可少的,并且可以使用RNaseZap TM 清洁表面。 RNA将用于微阵列分析和cDNA合成。
    3. 期望收益率> 4μgRNA/样品
    1. 从各自的小鼠样本中解剖TA肌肉或感兴趣的组织(图1A-1D)。
    2. 称取TA肌肉,在液氮中快速冷冻,并在1.5ml微量离心管中每50-100mg组织加入1ml TRIzol TM 试剂。
    3. 使用组织匀浆器将组织均质化2-3秒,将样品置于冰上以确保其不会过热。应将样品均质化直至看不到清晰的组织片,大约2-4次。
    4. 可选:通过在4℃和12,000 x g 离心5分钟澄清裂解物,并将上清液转移至新的1.5ml微量离心管(大约体积~800μl)。
    5. 在室温下孵育样品5分钟。
    6. 每1ml使用的TRIzol TM 试剂加入0.2ml氯仿,剧烈摇动或涡旋15秒。然后在室温下孵育样品3分钟。
    7. 在4℃下离心15分钟并在12,000 x g 中分离各相。
    8. 使用1 ml移液管(体积~400-600μl)将含有RNA的水相(透明,上图2)转移到新管中,确保不转移任何间期或酚 - 氯仿相。 />

      图2. TRIzol TM 试剂RNA提取后的相分离图像。水相(灰色)含有RNA,应小心转移到新管中进行下游分离。 br />
    9. 每1ml TRIzol TM 试剂加入0.5ml异丙醇,用于含有来自步骤A8的RNA的水相,并在室温下孵育10分钟。
    10. 将样品在4°C和12,000 x g 离心10分钟以沉淀RNA(RNA应在管底部形成白色颗粒)。
    11. 丢弃上清液,小心不要打扰颗粒。
    12. 每1ml用于洗涤RNA沉淀的TRIzol TM 试剂将沉淀重悬于1ml 75%乙醇中。
    13. 轻轻摇动样品,在4°C和7,500 x g 下离心5分钟。
    14. 小心丢弃上清液,尽可能多地去除乙醇,不要打扰沉淀。
    15. 风干颗粒在室温下最多5-10分钟。确保乙醇已蒸发,但不要让颗粒干燥太久,因为残留的乙醇和过度干燥都会影响RNA质量。 
    16. 将沉淀重悬于50μl不含RNase的水中(如果用于下游微阵列分析则势在必行)。
    17. 使用NanoDrop分光光度计测量浓度和A 260 / A 280 的比例。
      注意:
      1. 1毫克的样品应该产生大约1微克的RNA。
      2. 比率A 260 / A 280 下游应用需要 1.85-1.95的范围,应使用安捷伦生物分析仪检查RNA样品质量。
      3. 在Agilent生物分析仪上测定RNA样品的质量应该产生对应于18S和28S核糖体亚基的两个大峰(Peterson等,2009)。优质和劣质RNA的例子如图3所示。


        图3.使用Agilent BioAnalyzer测定的完整(理想),部分降解和降解RNA样品的图像。上图清楚地显示了两个峰,分别对应18S和28S核糖体亚基,一个小的峰值到尖峰控制和其他平滑的线条。中图和下图表示降解的RNA,其中18S和28S峰可能是或可能不是清楚的,并且通常在多个较小的峰之前(指示降解的RNA)。 x轴和y轴分别是核苷酸和荧光。

  2. 转录组微阵列分析
    微阵列方法和分析改编自制造商的手册(安捷伦,基于双色微阵列的基因表达分析 - 低输入快速放大器标记) 
    1. 制备Spike A Mix和Spike B Mix作为阳性对照,所有程序都应在无RNase的环境中进行,以确保RNA的稳定性,以下步骤可在1.5 ml微量离心管中进行。
      1. 涡旋和加热Spike A和B在到达时在37℃下混合5分钟并短暂离心。
      2. 稀释Spike A Mix(例如)通过向38μl稀释缓冲液中加入2μlSpikeA Mix制备第一次稀释液,混合并短暂旋转(稀释度为1:20)。
      3. 通过向78μl稀释缓冲液中加入2μl稀释溶液稀释来自步骤B1b的溶液,混合并短暂旋转(稀释度为1:40)。
      4. 通过向30μl稀释缓冲液中加入2μl稀释溶液稀释来自步骤B1c的溶液,混合并短暂旋转(稀释度为1:16)。
      5. 通过向28μl稀释缓冲液中加入4μl稀释溶液稀释来自步骤B1d的溶液,混合并短暂旋转(稀释度为1:8)。
      6. 将2μl最终稀释溶液(最终稀释度为1:102,400)加入25-100ng样品RNA(体积不应超过3.5μl)。
      7. 重复针对其他RNA样品的Spike B Mix(即,来自野生型样品的RNA与来自实验样品的RNA)并进行标记反应。
    2. 标记反应,纯化和定量荧光标记的互补RNA(cRNA)
      注意:对于Spike A,使用Cyanine 3-CTP和Spike B Cyanine 5-CTP染料进行制备,否则两种样品的处理方式相同。在开始步骤B2a-B2d(下面)之前,将水浴或加热块设置为65°C和80°C。 
      1. 通过将1.8μlT7引物与每次反应的1μl无核酸酶水组合来制备T7引物混合物。
      2. 向每个试管中加入1.8μlT7引物混合物,在65°C下孵育10分钟,每隔几分钟轻轻摇动试管。
      3. 从热量中移除反应并置于冰上5分钟。
      4. 同时,将5x第一链缓冲液在80°C预热3-4分钟,涡旋并旋转,使缓冲液组分完全重悬。
      5. 在1.5 ml微量离心管中将下列cDNA反应物装配在冰上(根据反应次数按比例放大,过量反应一次,以纠正移液错误):

      6. 向含有RNA + Spike Mix(最终体积为10μl)的每个管中加入4.7μlcDNA反应,通过移液混合并短暂旋转。
      7. 使用前预热至42°C和70°C 10分钟,以确保温度达到所需的温度(即,42°C和70°C)。 
      8. 在40℃下孵育反应2小时,然后在70℃加热灭活反应15分钟(偶尔摇动管或使用循环水浴)。
      9. 将样品置于冰上5分钟,同时按如下方法制备转录主混合物反应(根据反应次数按比例放大)以扩增和荧光标记RNA:

      10. 向Spike A Mix或Spike B Mix中加入6μl转录反应(每个反应的最终体积,现为16μl),上下移液混合,在40°C下孵育2小时。
      11. 使用RNeasy Mini Kit,从每个反应中纯化cRNA(根据制造商的说明总结下面的方案)
        1. 用无核酸酶水将cRNA反应体积调至100μl。
        2. 加入350μlRLT并充分混合。
        3. 加入250μl100%冰冷乙醇,轻轻吹打混合。
        4. 将反应物转移至离心柱,置于收集管中并在16,000 x g 和4℃下离心30秒。
        5. 弃去流过物并向柱中加入500μlRPE缓冲液,以16,000 x g和4°C离心30秒。
        6. 重复步骤B2j-B2k v,但在16,000 x g 和4℃下离心60秒。
        7. 将柱置于新鲜的收集管中并在16,000 x g 和4℃下离心30秒以除去并保留缓冲液。
        8. 将柱置于1.5ml微量离心管中,并向柱中加入30μl不含RNase的水。
        9. 在室温下孵育1-2分钟,然后在16,000 x g 和4°C下离心30秒。
        10. 可选:用洗脱液重新洗脱以提高产量。
        11. 使用NanoDrop TM ND-1000 UV-VIS分光光度计3.2.1或更高版本测量RNA浓度和质量。
          1)
          使用“微阵列测量”选项卡,选择RNA-40作为样品类型。
          2)
          记录花青3或5浓度,RNA吸光度比和cRNA浓度(RNA吸光度比[260/280 nm]应为1.9±0.04,如果使用2-pack格式,cRNA浓度应至少产生1.875μg)。
    3. 样品和探针的杂交
      1. 10x阻断剂的制备:向10x基因表达阻断剂(随试剂盒提供)中加入1,250μl不含核酸酶的水,轻轻涡旋以完全溶解粉末。
        注意:如果粉末不易溶解,则在37°C下加热阻滞剂4-5分钟。
      2. 将下列反应装入1.5 ml微量离心管中。
        注意:下面的反应和随后的反应是针对双组装微阵列,对于1包装,4包装或8包装,请参考安捷伦基于双色微阵列的基因表达分析方案。

      3. 在60°C孵育30分钟,然后将反应置于冰上。
      4. 加入等体积的2x Hi-RPM杂交缓冲液以停止反应并充分混合,小心不要引入任何气泡。
      5. 简单地将样品旋转并置于冰上以立即下游使用。
      6. 将垫片滑入Agilent SureHyb(图4B和4C)腔室底座,标签朝上。
      7. 从左到右缓慢地将240μl样品加到垫圈上,注意不要引入任何气泡。
      8. 在未使用的任何孔中,从2x Hi-RPM杂交缓冲液中制备1x溶液。
      9. 将载玻片放置在“活动”侧,使安捷伦标记的条形码朝下,数字条形码朝上。
      10. 将SureHyb腔盖放在载玻片上,夹紧两片并拧紧。
      11. 仔细检查没有固定的气泡,如果需要,可以轻敲表面以便移除。
        注意:气泡可能是伪影的来源,因为它们可能会影响信号强度。
      12. 将腔室加载到杂交炉中的转子架上,将其设置为以10rpm旋转并在65℃下杂交17小时。

    4. 洗涤微阵列载玻片
      1. 为了制备洗涤缓冲液,从容器中取出外盖和内盖,并用移液管将2ml Triton X-102加入基因表达洗涤缓冲液1和2中。
      2. 通过倒置进行混合,并使用随套件提供的套管替换原始的外帽和内帽。
      3. 预热基因表达洗涤缓冲液2至37℃,然后进行洗涤阵列。
      4. 使用前清洗染色皿如下(重复2次):
        1. 将载玻片架和搅拌棒加入染色皿中,并用100%异丙醇填充培养皿(图4A)。
        2. 打开磁力搅拌板,洗涤5分钟。
        3. 用Milli-Q水冲洗染色皿多次。


          图4.微阵列洗涤皿,SureHyb室和载玻片的图像。 A.带有金属搅拌棒(左)和载玻片架的洗涤皿的图像。 B.组装SureHyb室,盖子和夹子。 C. SureHyb装配套件的各个部件。顶部是腔室,微阵列载玻片位于其顶部,盖子(中间)轻轻放置在顶部。然后使用夹具(底部)确保滑块和腔室紧密地保持在一起。 D.微阵列载玻片的图像,可见条形码清楚地表示“安捷伦”并且用于正确定向。

      5. 准备染色盘如下
        1. 用基因表达洗涤缓冲液1填充载玻片染色皿#1。
        2. 将载玻片架放入载玻片染色皿#2中,加入磁力搅拌棒,填充基因表达洗涤缓冲液1并置于磁性板上。
        3. 将培养皿#3放在搅拌板上,加入搅拌棒,并在使用前立即填充预热基因表达洗涤缓冲液2。
      6. 从旋转培养箱中取出杂交室并记下杂交过程中可能形成的任何气泡。
      7. 通过将杂交室放置在平坦表面上来拆卸杂交室,移除阵列垫圈,同时保持数字条形码朝上并立即将其浸没在载玻片#1中。
      8. 将阵列垫片夹层浸入水中,用镊子撬开夹层,让垫圈滑到盘子的底部。
      9. 取下载玻片并将其放入载玻片#2的载玻片架中,小心只要触摸数字条形码上的滑块或薄边缘。
      10. 对剩余的幻灯片重复这些步骤。
      11. 将载玻片在磁力搅拌板上孵育1分钟。
      12. 将载玻片架添加到载玻片染色皿#3中并在磁力搅拌板上孵育1分钟。
      13. 慢慢小心地取下滑动架并将滑块放在载玻片架上。
        1. 将没有条形码标签的幻灯片添加到边缘。
        2. 活性微阵列表面应朝向滑盖。
        3. 关闭塑料盖。
      14. 继续扫描幻灯片。

    5. 扫描微阵列载玻片,特征选择和数据收集
      1. 将包含载玻片的载玻片架放入扫描仪盒中。
      2. 选择“AgilentG3_HiSen_GX_2color”协议。
      3. 点击“开始扫描”。
      4. 打开安捷伦特征提取(FE)并将要提取的图像添加到FE项目(项目的默认设置)。
        注意:可能需要手动网格映射。
      5. 通过文件>将特征提取项目另存为.fep。另存为。
      6. 选择项目>开始提取。

  3. 验证微阵列结果
    1. 用于实时定量PCR(qPCR)的基因特异性引物设计:选择扩增子大小,引物和模板对于生成可重现的数据是必不可少的,这些数据可以准确地确定微阵列的结果是否得到验证
      注意:预先验证的基因表达分析可以从各种供应商处购买,因此不需要优化。对于不可用或以前未经过验证的基因,应评估引物效率(见下文):
      1. 根据微阵列结果选择基因。应根据在不同条件下显示出显着变化的基因来选择候选基因,并且应选择不显示样品间表达的任何变化的参考基因(后者的实例包括18S rRNA或β-肌动蛋白)。
      2. 引物靶向的扩增子应大致相等(不大于0.6kb),靶位点的二级结构可使用核酸折叠软件如OligoAnalyzer 3.1(IDT)测定,因为高度结构化的序列可影响qPCR效率和结果。
      3. 引物靶位点应通过 in silico PCR工具(如NCBI BLAST或UCSC Genome Browser)进行分析,以确定特异性。
      4. 每种底漆应具有大致相同的熔化温度,但确切的理想退火温度必须通过实验确定。
    2. cDNA合成
      1. 需要在微阵列分析中测定来自组织样品的1至5微克模板RNA(在此使用生物和技术重复;前者是在相同条件下的另一个小鼠样品,后者是用于微阵列分析的相同RNA)和质量应在cDNA合成之前确定。
      2. 从M-MLV RT试剂盒中解冻试剂,涡旋并短暂离心。
      3. 按以下顺序在冰上组装以下反应:

      4. 轻轻摇动PCR管以混合内容物,短暂离心并在65℃下孵育5分钟,然后立即置于冰上。
      5. 按以下顺序将以下组分添加到反应中:

      6. 通过上下吸移混合反应,并在25℃下孵育10分钟。
      7. 在37°C孵育反应50分钟。
      8. 加热使反应在70℃下反应15分钟。
      9. 得到的cDNA可立即用于实时PCR分析或储存在-20°C。

    3. 候选靶基因的实时qPCR
      注意:设置生物和技术重复以及不含模板的阴性对照很重要。 3个生物学重复(即来自三个不同小鼠的三个RNA / cDNA样品),每个样品建议3个技术重复(即,使用相同的RNA产生cDNA)。
      1. 引物应在无核酸酶的水中再水合,并以10μM的储备浓度储存。
      2. 在冰上解冻试剂并在冰上准备以下反应(将最终反应体积乘以计划的PCR反应数加上[以解释移液错误],创建可混合到Roche LightCycler 480板中的主混合物)。 />
      3. 一般实时定量PCR循环仪设置。

      4. 然后可以分析C q 值的引物效率和随后的靶基因表达的倍数变化(不建议使用C q 值> 35)。 > 注意:需要超过35个循环的样品可能无法提供可靠的结果,并表明cDNA质量或反应效率很差。虽然一些非常低表达的基因可能在35-40之间产生C q 值,但在这些条件下,阴性对照必须在该周期数下不产生信号

数据分析

该协议的这一部分包括微阵列结果的分析和随后的基因集富集和独创性途径分析,以分别确定候选基因和富集的生物途径/过程。在候选基因选择之后,进行实时PCR分析以验证候选者(关于工作流程的概述,参见图5)。


图5.微阵列分析工作流程概述从感兴趣的组织样本(或细胞)中分离RNA。在进行cRNA的产生,杂交和数据采集之前确定RNA的质量。微阵列数据的分析由Agilent软件执行。从那里,使用Gene Set Enrichment Analysis软件产生在测定样品中显着富集的基因。微阵列数据还将用于Ingenuity途径分析(IPA)(并且可以用于基因本体论[GO]分析)以揭示在目标样品中富集的途径或生物过程。

  1. 微阵列结果分析
    标准化,基因比对和调用(关联基因表达水平)和评估基因表达变化的基因表达变化。
    1. 下载GeneSpring软件以执行统计分析和打开软件。
    2. 创建一个新项目,从特征提取器(FE)加载文本文件,然后单击“下一步”,将软件设置保持为默认值。
    3. 单击“完成”后,数据将上传到GeneSpring。
    4. 分配'实验分组'然后'创建解释',并选择适当的实验分组。
    5. 统计分析
      1. 使用软件执行方差分析(ANOVA),选择Tukey事后检验和相应的配对选项(取决于样品)。
      2. 如果比较野生型和敲除样本,执行学生 t - 测试(通常,执行的统计测试将取决于样本)。
    6. 倍数变化分析:消除不符合1.5倍变化的探针。
      1. 选择“折叠 - 更改”,使用与ANOVA相同的解释。
      2. 将倍数变化调整为1.5并确定有多少探针符合此标准。
      3. 选择“完成”后,应显示探针列表。
    7. 将表达增加或减少的探针组合成一个excel表,用于独创性通路分析。

  2. Ingenuity途径分析(IPA)
    IPA用于确定基于微阵列结果可在样品间改变的途径。
    1. 通过 www.ingenuity.com/products/ipa 登录IPA。
    2. 将从GeneSpring保存的excel文件上传到软件,选择“Agilent”和相应的识别类型(即,适当的种类)。
    3. 探针样品应为'ID',logFoldChange应为'观察1'。
    4. 单击“继续”,然后使用“分析就绪”列表选择“运行分析”。
    5. 然后将出现基因本体信息,并可以分析样品之间的途径富集。

  3. 基因集富集分析(GSEA)
    GSEA是对某些基因组中生物样品中基因表达差异是否达到统计学显着性的计算评估。这需要输入微阵列数据结果和GSEA参考数据集的选择(Subramanian et al。,2005)。
    1. 应对微阵列分析的结果进行过滤,得到一组基因,这些基因在实验样品中的表达变化≥1.5倍,达到显着性水平,校正的 P 值≤0.05。
    2. 下载GSEA软件并按照手册的说明进行安装(适用于R和Java)。
    3. 确定应进行分析的参考基因组:例如,对于Bi 等人(2016a),将人类DMD基因表达数据集与健康人体肌肉进行比较。选择是为了理解结果对小鼠模型的适用性(NCBI数据集GDS3027)。
    4. 包含来自步骤C1的表达数据的微阵列结果应转换为GCT文件类型:有关如何将文件转换为GSEA所需类型的详细信息,请参见 GenePattern:文件格式指南。
    5. 还必须生成包含要分析的参考数据集的基因集数据库文件以及用于输入GSEA的样本表型文件(以及用于存储输出结果的空目录)
      1. 基因集数据库文件(.gmt)格式详细信息可以在 GenePattern:文件格式指南。
      2. 样本表格文件(.cls)格式详细信息可以在 GenePattern:文件格式指南。
    6. 对于Bi et al。(2016a和2016b)中使用的分析,在调用GSEA时使用了默认设置。
      注意:但是,这些可以根据需要进行更改;有关详细信息,请参阅源代码文档。
    7. GSEA结果和图形分析
      1. 输出目录应包含GSEA摘要结果文件;在继续之前确定参数是否满足指定值(理想值可以在Subramanian et al。,2005中找到)。
      2. GSEA R包中包含GSEA.Analyze.Sets,它生成输入数据图,请参阅特定参数的源代码文档。

  4. 实时PCR结果分析(Bustin et al。,2009)(用于验证微阵列和GSEA发现的靶基因)
    1. 入门效率
      1. 应评估用于实时PCR的所有引物的效率。
      2. 所有引物应以与参照相对应的相似效率扩增靶位点。引物扩增的效率可以通过生成校准曲线来确定。
        1. 简而言之,校准曲线可以通过将高质量RNA逆转录成cDNA来确定。然后将cDNA连续稀释10倍4-5倍(即,1:10,1:100,1:1,000,1:10,000),并在每次稀释时测定引物。&nbsp ;
        2. 绘制初始log cDNA浓度 vs C q 值

          C q = m(log(cDNA浓度)+ b

          其中,m =斜率,b = y-截距
        3. 确定PCR效率
          效率测量为10 -1 / slope -1 
    2. 结果分析
      1. ΔΔC q 用于确定与参考基因相比的表达变化。
        1. 将候选基因的C q 值标准化为参考基因(ΔC q )。
        2. 转换归一化ΔC q 指数(log 2 (ΔC q ))。
        3. 取技术重复的平均值(即,来自同一生物样品的三个单独反应)并确定标准偏差; Bustin et al。(2009)中提供的指南。
        4. 归一化平均结果和标准偏差参考基因。
        5. 然后,折叠变化(1-ΔΔC q )×100。
      2. 折叠变化方向性应与微阵列分析的结果一致。

笔记

  1. 使用实时定量PCR验证微阵列结果
    1. 使用针对靶基因的实时定量PCR验证微阵列结果应该在GSEA之前进行,因为必须确保从微阵列获得的结果可以通过替代方法再现(从而证实其生物学意义)。 
    2. 基因表达和实时定量PCR的所有阈值在上述方案中详述。同样,必须具有阳性和阴性对照以及生物学和技术重复以达到统计学显着性(通常,至少三次重复/样品,但应使用功效分析来确定样品大小)。例如,生成cDNA时的阴性对照反应应该用于随后的实时定量PCR,以确认没有污染物质。 
    3. 微阵列结果可以通过使用用于微阵列分析的相同RNA的实时定量PCR来验证。但是,应进行功效分析以确定每个实验的适当样本量,并且除非另有说明,否则具有双尾分布的学生 t - 测试可用于分析结果。 
  2. 使用基因本体论(GO)术语分析验证微阵列结果
    基因本体论(GO)术语分析也可以在微阵列产生的基因列表上进行;然而,GSEA为每个基因提供等级和权重,以便考虑样本中的相对表达水平,从而帮助研究人员识别候选基因。 GO术语分析不提供基因特异性信息,但GSEA和GO术语分析将产生在测定样品中显着富集的生物途径

致谢

NIH拨款R01CA212609和R01AR071649部分支持该研究。协议改编自下文参考文献中列出的Bi 等人(2016a和2016b)。

利益争夺

作者声明没有利益冲突或竞争利益。

伦理

涉及使用动物的所有程序均按照普渡大学动物护理和使用委员会(PACUC)提出的指南进行。

参考

  1. Amaratunga,D.,Göhlmann,H。和Peeters,P。J.(2007)。 3.05 - 微阵列。在:Taylor,JB和Triggle,DJ(编辑) 。综合药物化学II。 Elsevier ,87-106。
  2. Bi,P.,Yue,F.,Karki,A.,Castro,B.,Wirbisky,SE,Wang,C.,Durkes,A.,Elzey,BD,Andrisani,OM,Bidwell,CA,Freeman,JL, Konieczny,SF和Kuang,S。(2016b)。 Notch激活可促进小鼠脂肪细胞去分化和肿瘤发生转化。 J Exp Med 213(10):2019-2037。
  3. Bi,P.,Yue,F.,Sato,Y.,Wirbisky,S.,Liu,W.,Shan,T.,Wen,Y.,Zhou,D.,Freeman,J。and Kuang,S。( 2016a)。 在骨骼肌生成过程中Notch激活的阶段特异性影响。 Elife 5:e17355。
  4. Bustin,SA,Benes,V.,Garson,JA,Hellemans,J.,Huggett,J.,Kubista,M.,Mueller,R.,Nolan,T.,Pfaffl,MW,Shipley,GL,Vandesompele,J。和Wittwer,CT(2009年)。 MIQE指南:发布定量实时PCR实验的最低限度信息。 Clin Chem 55(4):611-622。
  5. Peterson,S.M。和Freeman,J.L。(2009)。 从胚胎斑马鱼中分离RNA并进行基因表达分析的cDNA合成。 J Vis Exp (30):1470。
  6. 阅读,J。和Brenner,S。(2001)。 Microarray Techonlogy。在:Brenner,S。和Miller,JH(编辑) 。 遗传学百科全书。学术出版社1191。
  7. Wang,Z.,Gerstein,M。和Snyder,M。(2009)。 RNA-Seq:转录组学的革命性工具。 Nat Rev Genet 10(1):57-63。
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Copyright Oprescu et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Oprescu, S. N., Horzmann, K. A., Yue, F., Freeman, J. L. and Kuang, S. (2018). Microarray, IPA and GSEA Analysis in Mice Models. Bio-protocol 8(17): e2999. DOI: 10.21769/BioProtoc.2999.
  2. Bi, P., Yue, F., Sato, Y., Wirbisky, S., Liu, W., Shan, T., Wen, Y., Zhou, D., Freeman, J. and Kuang, S. (2016a). Stage-specific effects of Notch activation during skeletal myogenesis. Elife 5: e17355.
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