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Microarray and qPCR support

Tecan offers cDNA generation kits for gene expression microarrays and qPCR. General information on sample preparation, library preparation and sequencing are provided below. For more information, please consult the User Guide for each product, or contact Tecan NGS Technical Support at techsupport-gn@tecan.com.

For information on using the TrueMethyl oxBS Module with EPIC arrays, contact Tecan NGS Technical Support.

 

RNA input recommendations

  • Total (All products) or poly A selected RNA (Ovation Pico, Ovation PicoSL and Ovation RNA Amp Systems only)
  • High quality, with an A260:A280 ratio of 1.8 or above, a A260:A230 of 2.0 and above, and a RIN score above 7
    Note: While high quality RNA will guarantee the best results, many of our kits will perform with degraded samples.
  • DNase treated. We recommend a DNase such as HL-dsDNase from ArcticZymes. Other suitable DNase treatments include:

 

RNA isolation

We recommend a column-based method, including:

 

For FFPE RNA isolation, we recommend a kit designed for FFPE samples, including:

 

Ensure that the elution volume/final RNA concentration is appropriate for the cDNA kit used. Tecan cDNA kits use input volumes of 5-10 µL.

Do not use carriers during RNA isolation. Organic methods such as TRIzol® Reagent should be subsequently followed with a column-based clean-up method.

 

cDNA clean-up

cDNA generated with Tecan cDNA Systems should be cleaned up using a column-based method such as:

Alternatively, an SPRI bead-based system such as Agencourt® RNAClean® XP or AMPure XP are suitable. If choosing a bead-based method, we recommend using a magnet compatible with 0.2 mL tubes, tube strips, or plates.

Note: Columns or beads for final cDNA cleanup must be purchased separately.

 

Preparation of cDNA for qPCR assays

We recommended that the amplified cDNA from Tecan’s cDNA generation kits be purified prior to use in real-time quantitative PCR reactions. Since different amplified cDNA samples may be variable in concentration, the purified products can be quantified and mass-normalized to ensure the cDNA inputs to qPCR are equal for all samples.

Amplified cDNA produced with these kits can be used successfully as template for qPCR systems including TaqMan® and SYBR™ Green.

Follow the recommendations for qPCR reagents, sample dilution and primer design as noted by Tecan product listed below. Other qPCR master mixes are likely compatible with the amplified cDNA produced with these kits but have not been thoroughly evaluated by Tecan.

 

  • Crescendo cDNA Synthesis for qPCR
    • The following reagents have been successfully used for qPCR:
      • TaqPath qPCR Master Mix, CG (Thermo Fisher Scientific, Cat. #A16245)
      • QuantiTect™ SYBR Green PCR Kit (QIAGEN, Cat. #204143)
      • iQ SYBR Green Supermix (BioRad, Cat. #170-8880)
    • Recommendations to achieve optimal results
      1. Dilute the amplified product
        • The appropriate input of amplified cDNA for use in qPCR reaction should be determined empirically.
        • Higher inputs of cDNA may be required for qPCR when starting with limited or degraded total RNA.
        • Higher inputs of cDNA may be required for qPCR when detecting low-expressed transcripts.
      2. Primer design
        • Primers may be designed at any position along a transcript since the Crescendo cDNA Synthesis for qPCR amplification process covers the whole transcriptome.
        • When starting with high quality RNA, we recommend using primers and probes designed with amplicon sizes of less than 200 nt.
        • When starting with degraded RNA, we recommend using primers and probes designed with amplicon sizes of less than 100 nt to compensate for the smaller cDNA fragments.
  • Ovation Pico WTA System V2 and Ovation PicoSL WTA System V2
    • The following reagents have been successfully used for qPCR
      • TaqMan: ABsolute qPCR Mix plus ROX (Thermo Fisher Scientific, Cat. #AB1136/B)
      • TaqMan Fast Universal PCR Master Mix 2x (Thermo Fisher Scientific, Cat. #4352042)
      • QuantiTect™ SYBR Green PCR Kit (QIAGEN, Cat. #204143)
      • iQ SYBR Green Supermix (BioRad, Cat. #170-8880)
      • FastStart SYBR Green Master (ROX) (Roche, Cat. #04 673 514 001)
      • Note: RT-PCR master mixes containing the enzyme Uracil N-Glycosylase (UNG) are not compatible with the Ovation Pico WTA System V2 or Ovation PicoSL WTA System V2.
    • Recommendations to achieve optimal results
      1. Dilute the SPIA cDNA
        After SPIA cDNA purification and quantification, dilute to an appropriate concentration for qPCR reaction. We recommend using 20 ng of SPIA cDNA in a 20 µL TaqMan reaction and 2 ng of cDNA for a 25 µL SYBR Green reaction. More or less cDNA may be required depending on the abundance of the targeted transcripts.
      2. Primer Design
        We recommend using primers and probes designed with amplicon sizes of less than 200 nt. Primers may be designed at any position along a transcript since the Ovation Pico WTA System V2 amplification covers the whole transcriptome.
  • Ovation RNA Amplification System V2
    The amplified cDNA generated from the Ovation RNA Amplification System V2 may be used directly in real time quantitative PCR reactions or purified first in order to quantify and normalize the input for qPCR reactions.
    • The following reagents have been successfully used for qPCR:
      • FastStart TaqMan Probe Master (Rox) (Roche, Cat. #04 673 476 001)
      • TaqMan: ABsolute qPCR Mix plus ROX (Thermo Fisher Scientific, Cat. #AB1136/B)
      • TaqMan Fast Universal PCR Master Mix 2x (Thermo Fisher Scientific, Cat. #4352042)
      • QuantiTect™ SYBR Green PCR Kit (QIAGEN, Cat. #204143)
      • iQ SYBR Green Supermix (BioRad, Cat. #170-8880)
      • FastStart SYBR Green Master (ROX) (Roche, Cat. #04 673 514 001)
      • Note: RT-PCR master mixes containing the enzyme Uracil N-Glycosylase (UNG) are not compatible with the Ovation RNA Amplification System V2.
    • Recommendations to achieve optimal results
      1. Dilute the SPIA cDNA
        • Using unpurified SPIA cDNA:
          We recommend diluting the unpurified cDNA before performing qPCR because inhibition has been observed with undiluted product. Dilute the amplified product 1:10 in nuclease-free water or in a buffer specified by the qPCR system manufacturer, and use 2 µL per 25 µL of qPCR reaction. Additional dilution may be required depending on the abundance of the targeted transcripts.
        • Using purified SPIA cDNA:
          Purification of the amplification products prior to qPCR is highly recommended, especially for SYBR Green assays. Purified cDNA is typically obtained at a concentration of 150 to 250 ng/µL. Dilute the cDNA to an appropriate level for use in qPCR reactions. Inputs in the range of 5 - 10 ng in a 25 µL qPCR reaction are typically appropriate, however it may be necessary to empirically optimize the input for low or high copy-number transcripts.
      2. Primer Design
        • The amplified cDNA produced using the Ovation RNA Amplification System V2 is generated from the 3´ ends of the mRNA. For best results, primer sets used in qPCR procedures using amplified cDNA should be within 1.5 Kb of the poly(A) tail.
  • Ovation FFPE WTA System
    • Tecan Genomics has successfully used the following reagents for qPCR:
      • TaqMan ABsolute qPCR Mix plus ROX (Thermo Fisher Scientific, Cat. #AB-1136/B)
      • TaqMan Fast Universal PCR Master Mix 2x (Thermo Fisher Scientific, Cat. #4352042)
      • QuantiTect™ SYBR Green PCR Kit (QIAGEN, Cat. #204143)
      • iQ SYBR Green Supermix (BioRad, Cat. #170-8880)
      • FastStart SYBR Green Master (ROX) (Roche, Cat. #04 673 514 001)
      • Note: RT-PCR master mixes containing the enzyme Uracil N-Glycosylase (UNG) are not compatible with the Ovation FFPE WTA System.
    • Recommendations to Achieve Optimal Results
      1. Dilute the SPIA cDNA
        After SPIA cDNA purification and quantification, dilute to an appropriate concentration for qPCR reaction. We recommend using 20 ng of SPIA cDNA in a 20 µL TaqMan reaction and 2 ng of cDNA for a 25 µL SYBR Green reaction. More or less cDNA may be required depending on the abundance of the targeted transcripts.
      2. Primer Design
        We recommend designing multiple assays across the length of the transcript since the starting FFPE RNA is likely to be highly degraded and some amplicons may not perform robustly. Primers and probes should be designed with as small an amplicon size as possible due to the degraded nature of the input RNA. Primers may be designed at any position along a transcript since the Ovation FFPE WTA System amplification covers the entire length of the transcript.

 

Fragmentation, Labeling, and Hybridization of cDNA for Microarrays

Tecan’s Encore Biotin Module is designed solely for use with cDNA prepared using following Tecan cDNA kits:

  • Ovation® RNA Amplification System V2 (Part No. 3100)
  • Ovation Whole Blood Solution (Part Nos. 3100 and 1300)
  • Ovation Pico WTA System V2 (Part No. 3302)
  • Ovation PicoSL WTA System V2 (Part No. 3312)*
  • Ovation FFPE WTA System (Part No. 3403)i

*Note: With the Ovation PicoSL System V2, use half the recommended volumes throughout the Encore Biotin Labeling protocol.

 

Fragmentation and labeling for GeneChip Arrays

Tecan amplification system
(Part No.)
cDNA input
per reaction*
Final HYB cocktail
concentration
Ovation FFPE WTA System (Part No. 3403) 4-5 μg 18–23 ng/μL
Ovation Pico WTA System V2 (Part No. 3302) 5 μg 23 ng/μL
Ovation PicoSL WTA System V2 (Part No. 3312) 2.5 μg** 23 ng/μL
Ovation RNA Amplification System V2 (Part No. 3100) 3.75 μg 17 ng/μL
Ovation Whole Blood Solution (Part No. 1300 & 3100) 4.4 μg 20 ng/μL

* All cDNA concentrations are assessed using the single-stranded DNA setting on a spectrophotometer (33 μg/mL/A260 as the constant).

** Requires performing half-volume Encore Biotin Module reactions. Refer to the appropriate Ovation PicoSL WTA System user guide for specific guidelines.

 

Hybridization of Labeled cDNA Target on GeneChip and Clariom Arrays

The tables below show recommendations for hybridization cocktail assembly and appropriate fluidics protocols for GeneChip®and Clariom Arrays. GeneChip™ Hybridization, Wash and Stain Kit must be purchased separately (Thermo Fisher Scientific P/N 900720, 901622).

Component Standard GeneChip Array, Clariom D Array (49 or 64 Format) GeneChip Midi Array (100 Format) GeneChip Mini Array, Clariom S Array (169 Format) Final Concentration
Fragmented, biotin-labeled amplified cDNA 50 μL 34 μL 25 μL Depends on sample type and amplification method
Control oligonucleotide B2 (3 nM) 3.7 μL 2.5 μL 1.9 μL 50 pM
20X Eukaryotic hybridization controls (bioB, bioC, bioD, cre) 11 μL 7.5 μL 5.5 μL 1.5, 5, 25 and 100 pM, respectively
2X Hybridization buffer 110 μL 75 μL 55 μL 1X
100% DMSO 22 μL 15 μL 11 μL 10%
Water 23.3 μL 16 μL 11.6 μL N/A
Total Volume 220 μL 150 μL 110 μL  
Array Loading Volume 200 μL 130 μL 90 μL  
Fluidics protocols
For 3’ arrays FS450_0004 FS450_0002    
For ST arrays and Clariom arrays FS450_0001
(Exon arrays)
  FS450_0007
(Gene arrays)
 

 

 

Component Clariom S HT (array plates) Vol for 1 array Clariom S HT (array plates ) Vol for 16-array plate Clariom S HT (array plates ) Vol for 24 array-plate Clariom S HT (array plates ) Vol for 96-array plate Final Concentration
Hybridization Master Mix          
5X WT Hyb Add1 20 µL 352 µL 528 µL 2112 µL 1X
3 nM Control Oligo B2 1 µL 17.6 µL 26.4 µL 105.6 µL 30 pM
20X Eukaryotic hybridization controls (bioB, bioC,bioD, cre) 5 µL 88 µL 132 µL 528 µL 1.5, 5, 25 and 100 pM, respectively
15X WT Hyb Add 4 6.7 µL 117.9 µL 176.9 µL 707.5 µL 1X
Nuclease-free water 2.3 µL 40.5 60.7 µL 242.9 µL  
Total Volume 35 µL 616 µL 924 µL 3696 µL  
Fragmented, biotin-labeled amplified cDNA 25 µL       depends on sample type and amplification method
2.5X WT Hyb Add 6 40 µL       1X
Total Volume 100 µL        

Overage has been included in these volumes.