Microarray and qPCR 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:
• Qiagen RNase-free DNase (included with the RNeasy Mini Kit and RNeasy Micro Kit.
• Roche RNase-free DNase followed by clean-up with Zymo RNA Clean & Concentrator 5 or Qiagen RNeasy MinElute Cleanup Kit.
• Column-based methods are not recommended as they may not provide sufficient removal of DNA.

RNA isolation

We recommend a column-based method, including:

• Norgen Biotek Total RNA Purification Kit
• Zymo Research QuickRNA TM Kits
• Arcturus PicoPure® RNA Isolation Kit
• Ambion PureLink® RNA Mini Kit
• Qiagen RNeasy Kits

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

• Norgen Biotek FFPE RNA Purification Kit
• Zymo Research Quick-RNA™ FFPE Kit
• Arcturus® Paradise® PLUS FFPE RNA Isolation Kit
• PureLink™ FFPE RNA Isolation Kit
• Qiagen RNeasy FFPE Kit

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:

• QIAGEN MinElute Reaction Cleanup Kit
• QIAGEN QIAquick PCR Purification Kit
• Zymo Research DNA Clean & Concentrator TM -25

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

* 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).

Overage has been included in these volumes.