Complete Whole Transcriptome Amplification Kit Protocol (WTA2)

Product Description
Components
Storage/Stability
Procedure
Troubleshooting Guide
Frequently Asked Questions
Materials
References
Publication

Product Description

WTA2, a Whole Transcriptome Amplification (WTA) method, allows for representative amplification of nanogram quantities of total RNA in less than 4 hours without 3'-bias. The resulting microgram quantities of product generated from tissue, cultured cells, formalin-fixed samples, or serum are suitable for downstream applications such as qPCR and microarray analyses. The Complete WTA Kit provides everything needed for amplification including the amplification enzyme.

The WTA process involves two steps. In the first step, sample RNA is reverse transcribed with primers composed of a semi-degenerate 3' end and a universal 5' end. As polymerization proceeds, displaced single strands serve as new templates for primer annealing and extension. The resultant cDNA library, composed of random, overlapping fragments flanked by universal end sequence, is then amplified by PCR with the universal primer to produce WTA product. Product size ranges from 100–1000 bases when amplifying intact RNA, and typically smaller for degraded RNA.

Components

Description	Catalog Number	10 RXN	50 RXN
Library Synthesis Buffer	L9418	25 µL	125 µL
Library Synthesis Solution	L9293	25 µL	125 µL
Library Synthesis Enzyme	L9543	20 µL	100 µL
Amplification Mix	A6731	375 µL	1.875 µL
10mM dNTP Mix	D7295	0.2 mL	0.5 mL
Nuclease-Free Water	W4502	5 mL	20 mL
Amplification Enzyme	A6856	37.5 µL	187.5 µL

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

Following this procedure should produce 25-40 µg of WTAmplicon, starting from 5-25 ng of high quality total RNA. Typically, 5- to 10-fold more input RNA is recommended when isolated from FFPE tissues. Reactions can be scaled up or down to accommodate preparation of necessary quantities of final product.

Library Synthesis Reaction

1. Thaw the Library Synthesis Buffer, Library Synthesis Solution, Library Synthesis Enzyme and Nuclease-Free Water. Mix Library Synthesis Buffer and Library Synthesis Solution thoroughly. Dissolve any precipitate in these solutions by briefly heating at 37 °C, with thorough mixing.

2. To at least 25 ng of total RNA (5 ng per 75 µL reaction, step 10) add:
a. 2.5 µL Library Synthesis Solution
b. Nuclease-Free Water to a total of 16.6 µL

Note: At least 250 ng of degraded total RNA, e.g., from FFPE tissues, is recommended per 375 µL reaction (50 ng per 75 µL reaction, step 10).

3. Mix and incubate in a thermocycler programmed for 70 °C for 5 minutes then 18 °C.

4. To the cooled-primed RNA immediately add the following (individually or premixed)
a. 2.5 µL Library Synthesis Buffer
b. 3.9 µL Water
c. 2 µL Library Synthesis Enzyme

5. Incubate in a thermal cycler using the following parameters:
18º C for 10 minutes
25º C for 10 minutes
37º C for 30 minutes
42º C for 10 minutes
70º C for 20 minutes
4º C

6. Consolidate any condensation by centrifugation and mixing.

Amplification Reaction

7. Thaw the Amplification Mix and 10mM dNTP Mix.

8. Prepare the following master mix
a. 301 µL Nuclease-Free Water
b. 37.5 µL Amplification Mix
c. 7.5 µL WTA dNTP Mix
d. 3.75 µL Amplification Enzyme

Note: For real-time PCR, deduct the volumes of a reference dye and 3.75 µL of a 1:1000 dilution (in Nuclease–Free Water) of SYBR^® Green stain from the water volume. Add SYBR^® Green dilution to master mix immediately before dispensing. Prepare a fresh dilution for each experiment,

9. Add the entire Library Synthesis reaction from Step 6 (25 µL) to the master mix solution from Step 8 and mix.

10. Divide the above (step 9) into five 75 µL reactions. (A reaction volume of < 75 µL, for the last aliquot, is not critical). Incubate in a thermal cycler using the following parameters:
94° C for 2 minutes.
17 cycles x (94° C for 30 seconds, 70° C for 5 minutes)

Note: The optimal number of amplification cycles varies with template amount and quality. Seventeen cycles is recommended for 5 ng of high quality RNA or 50 ng of FFPE RNA. Due to variations in the level of degradation (e.g., RNAs isolated from FFPE tissues), some RNA samples may require higher input quantities and/or more cycles. Optimal cycle number is achieved by proceeding 2–3 cycles beyond the amplification “plateau”, as observed with real-time quantitative PCR.

11. 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.

12. For removal of residual primers and nucleotides, use any standard PCR purification kit or equivalent methods for purification of double and single-stranded DNA.

13. 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 present following amplification.

PicoGreen and SYBR are registered trademarks of Molecular Probes. Inc.

Troubleshooting Guide

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
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.

Can WTA2 be used to amplify archival fixed tissue or degraded samples?
Yes, the WTA kit effectively amplifies degraded RNA, including formalin-fixed and paraffin-embedded samples. However, to acceptably amplify the final product, degraded samples require more starting material, but no more than 300 ng should be used.

What materials are provided with the WGA2 kit?
The WGA2 provides all necessary buffers, primers and enzymes for first strand synthesis, second strand synthesis and cDNA library amplification.

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.

Is the WTA2 system 3 prime biased?
No, in the system, quasi-random WTA primers with universal ends are used for synthesis of cDNA products. This enables the amplification of highly degraded RNA in which much of the amplifiable sequence has become separated from the poly-A sequence. qPCR and microarray testing of both degraded and intact RNA vs 3' biased methods have been successful.

Where in my target sequence can I design my qPCR primers?
The system WTA2 includes random priming and therefore primers can be designed at any location within the mRNA. In order to avoid qPCR interference from possible genomic DNA contamination, we recommend treating your RNA with DNase and designing your amplicons to span an intron. We strongly recommend designing your assays for multiple locations across the transcript since the starting FFPE RNA is likely to be highly degraded.

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.

References

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8. Lim YW, Schmieder R, Haynes M, Willner D, Furlan M, Youle M, Abbott K, Edwards R, Evangelista J, Conrad D, et al. 2013. Metagenomics and metatranscriptomics: Windows on CF-associated viral and microbial communities. Journal of Cystic Fibrosis. 12(2):154-164. https://doi.org/10.1016/j.jcf.2012.07.009

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