TransPlex® Whole Transcriptome Amplification Kit Protocol

Product No. WTA1

Product Description

TransPlex, a Whole Transcriptome Amplification (WTA) method, allows for representative amplification of nanogram quantities of total RNA in less than 4 hours without 3'-bias. Microgram quantities of amplification product generated from tissue, cultured cells, formalin-fixed samples, or serum are suitable for downstream applications such as qPCR and microarray analyses.

The WTA process involves two steps. In the first step, sample RNA is reverse transcribed with non-self-complementary primers composed of a quasi-random 3' end and a universal 5' end. As polymerization proceeds, displaced single strands serve as new templates for primer annealing and extension. The resultant Omniplex® cDNA library, composed of random, overlapping 100–1000 base fragments flanked by universal end sequence, is then amplified by PCR with the universal primer to produce WTA product.

Components

Description Catalog Number 50 RXN
WTA Library Synthesis Buffer
L7168 135 µL
WTA Library Stabilization Solution
L7043 135 µL
WTA Library Synthesis Enzyme
L6918 60 µL
WTA Amplification Master Mix
A0356
2 x 1.0 mL
10 mM dNTP Mix D7295 500 µL
Water, Molecular Biology Reagent W4502 4 x 5.0 mL

*Some Taq DNA polymerases can lead to the formation of product in no-template controls. This no-template product will not contain genes of interest if probed using PCR or hybridization techniques.
**Nuclease-free

Storage/Stability

All components should be stored at –20 °C. When thawed for use, components should be kept on ice. Stability of the WTA Library Synthesis Enzyme will be affected if stored warmer than –20 °C or allowed to remain for long periods at temperatures over 4 °C.  RNA sample (not included) should be thawed on ice.

Procedure

Library Preparation

  1. Thaw WTA Library Synthesis Buffer and WTA Library Stabilization Solution on ice and mix thoroughly. Dissolve any precipitate in these solutions by briefly heating at 37 °C and mixing thoroughly.
  2. To 5–300 ng of total RNA, add, individually or pre-mixed, the following:
        2.5 µL WTA Library Synthesis Buffer
        2.5 µL WTA Library Stabilization Solution
        Nuclease-free water for a total volume of 24 µL.
  3. Mix by pipetting and incubate at 70 °C for 5 minutes.
  4. Cool reaction immediately on ice and spin down any condensation by centrifugation.
  5. Add 1 µL of WTA Library Synthesis Enzyme and mix by pipetting.
  6. Incubate in thermal cycler using the following parameters:
        24 °C for 15 minutes
        42 °C for 2 hours
        95 °C for 5 minutes
  7. Chill reaction immediately on ice. Spin down any condensation by centrifugation.

Amplification

  1. Thaw WTA Amplification Master Mix and dNTP Mix on ice and mix thoroughly.  
  2. Prepare the following WTA Amplification Mix:*
        300 µL Water, Molecular Biology Reagent
        37.5 µL WTA Amplification Master Mix
        7.5 µL dNTP Mix
        12.5 units of antibody inactivated hot-start Taq DNA Polymerase

*For real-time PCR, include a reference dye as necessary and 3.75 µL of a 1:1000 dilution of SYBR® Green stain.

  1. Divide the library, placing 5 µL aliquots in individual tubes or wells.
  2. Add 70 µL of WTA Amplification Mix to each aliquot and mix well. Incubate in thermal cycler using the following parameters:
        95 °C for 3 minutes.
        17 cycles* x (94 °C for 20 seconds, 65 °C for 5 minutes)

*Optimal cycle number varies with template amount and quality. 17 cycles is recommended for 5 ng of high quality RNA. Optimal cycle number is achieved by proceeding 2–3 cycles into the amplification “plateau”.

  1. After cycling is complete, maintain the reactions at 4 °C or store at –20 °C until ready for analysis or purification. The stability of WTA DNA is equivalent to genomic DNA stored under the same conditions.
  2. For removal of residual primers and nucleotides, use any standard PCR purification kit or equivalent methods for purification of double and single-stranded DNA.  
  3. Purified DNA is quantified by measuring absorbance. 1 A260 unit is equivalent to 50 ng/µL DNA. Measurement techniques such as PicoGreen® dye will often underestimate the actual WTA DNA yield, since single stranded DNA may be generated during amplification

Troubleshooting

Observation Potential Cause
Recommended Solution
Low yield







Sample RNA quality (degraded or impure)



Titrate input RNA quantity up to 300 ng
Evaluate different RNA preparation methods
Increase PCR cycles
Monitor amplifications on real-time instrument to determine optimal PCR cycle
Pool multiple reaction product of degraded or impure samples
Quantified using PicoGreen Determine yield by UV absorbance
Didn't purify single-stranded or small products
Use a kit that purifies double and single-stranded DNA
Use a kit capable of purifying 100 bp PCR products
Didn't use a hot start Taq DNA Polymerase Use an antibody inactivated hot-start Taq DNA Polymerase
Rare transcripts not efficiently incorporated during library amplification Insufficient RNA input Increase RNA quantity

Frequently Asked Questions

What type of RNA may be used?

RNA can be isolated using standard methods or kits; RNA from numerous source materials may be used including blood, tissue biopsy, cultured cells and fixed or frozen tissues. Non-human sources of RNA may also be used such as animals, plants, or microorganisms.

How much RNA is required to successfully perform WTA amplification?

For the most robust performance there should be at least 50 ng of RNA at a concentration of >5 ng/µl in TE, samples of RNA containing < 5 ng are useable. The RNA can be single-stranded or double-stranded and should have a molecular weight of at least 300 bases.

How can I optimize amplification yields?

Optimal PCR cycle numbers might vary with template amount and quality. 17 cycles are recommended for 5 ng library aliquots of high quality RNA. If using a real-time system, the optimal cycle number is defined as the last cycle of a 2-3 cycle "plateau" phase in which the relative fluorescence unit stays constant.

Once library amplification is complete how should the samples be purified?

Upon completion of library amplification the cDNA should be purified to remove residual primers and nucleotides that may interfere with downstream applications. We recommend the Sigma-Aldrich® GenElute PCR DNA Purification Kit (NA1020) for the purification of single-stranded and/or double stranded amplification products from other reaction components such as excess primers, nucleotides or polymerases.

I need to quantify my WTA product, what is the preferred method?

UV absorbance (A260) should be used to quantify purified products using the conversion of 1 OD = 50 µg/ml. PicoGreen® should not be used for quantification because it cannot efficiently detect single-stranded products and will underestimate the DNA yield. Each aliquot of library generated from high quality human total RNA will generate between 4 to 8 µg of WTA product. If electrophoresed, the product appears as a smear with a size distribution of 0.2 kb to 2 kb on a 0.8% agarose gel. Yield and size distribution of products may vary depending on the integrity and purity of the sample RNA.

What are the downstream applications of WTA products?

WTA amplification creates a cDNA library of the RNA template. Applications such as qPCR, traditional cloning (TOPO TA Cloning®), micro array may be performed.

Can I adjust the protocol?

Yes, the protocol is optimized to create microgram quantities of cDNA for microarray analysis. Scaling back the reaction volumes will not impact the quality of the results, only the quantity of final product.

I want to clone the cDNA library—what are suggestions?

The cDNA product is similar to any PCR product. A TOPO TA cloning method is suggested. Ensure a dilution is set-up to achieve the optimal cloning results.

Labeling Protocols for Microarrays

  • Affymetrix WTA Application Note (378 Kb PDF)
  • Agilent WTA Application Note (840 Kb PDF)
  • NimbleGen WTA Application Note (435 Kb PDF)
  • Illumina WGA Application Note (202 Kb PDF)

Materials

     

 References

  1. Yokobayashi et al PRC1 coordinates timing of sexual differentiation of female primordial germ cells.
  2. Pan X et al Two methods for full-length RNA sequencing for low quantities of cells and single. Audio and Electroacoustics Newsletter, IEEE 2013-01-08 (2013)
  3. Vargas, M et al Aromatic amino acids required for pili conductivity and long-range extracellular electron transport in Geobacter sulfurreducens. (2013)
  4. Benaypun, B.A. et al Adult ovarian granulosa cell tumor transcriptomics: prevalence of FOXL2 target genes misregulation gives insights into the pathogenic mechanism of the p.Cys134Trp somatic mutation. Oncogene, 2012-07-16 (2012)
  5. Planamente, S.S et al  Structural basis for selective GABA binding in bacterial pathogens, Molecular Microbiology 2012-12-01 
  6. Parker,JK et al  Amplification of viral RNA from drinking water using TransPlex™ whole-transcriptome amplification, Journal of Applied Microbiology 2011-07-01
  7. Nagy, Z.B, et al.  Real-time polymerase chain reaction-based exponential sample amplification for microarray gene expression profiling. Anal. Biochem, 337, 76-83.(2005)
  8. Klur, S, et al.  Evaluation of procedures for amplification of small–size samples for hybridization on microarrays. Genomics, 83, 508 17 (2004).
  9. Iscove, N.N., et al. Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA. Nat. Biotechnol, 20, 940-3 (2002).
  10. Hertzberg, M, et al. cDNA microarray analysis of small plant tissue samples using a cDNA tag target amplification protocol. Plant J, 25, 585-91 (2001).

 

Agencourt is a registered trademark of Beckman Coulter, Inc.
GenElute is a trademark of Sigma-Aldrich Co. LLC.
NanoDrop is a registered trademark of Thermo Scientific, Inc.
Sybr is a registered trademark of Molecular Probes. Inc.
TransPlex and GenomePlex are registered trademarks of Rubicon Genomics, Inc.
TRI Reagent is a registered trademark of Molecular Research Center, Inc.

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