The SeqPlex RNA Amplification kit provides a method for amplification of total RNA or isolated mRNA prior to entry into the workflows of the commonly used deep sequencing platforms. Microgram quantities of double-stranded cDNA are generated from low nanogram to picogram quantities of total RNA in eight hours or less. The SeqPlex RNA Amplification Kit amplifies RNA (including non-polyA-tailed RNA) isolated from tissue, cultured cells, formalin-fixed samples, or serum while maintaining patterns of differential expression found in the unamplified sample.
Preparation for sequencing involves three steps. First, sample RNA is reverse transcribed with primers having a semi-degenerate 3′-end and a defined universal 5′-end. As DNA polymerization proceeds, displaced single strands serve as new templates for primer annealing and extension. Next, the resultant double-stranded cDNA library, composed of random, overlapping fragments flanked by a single universal primer sequence is amplified under optimized PCR conditions to produce a WTA-product (whole transcriptome amplification). With nanogram quantities of intact input RNA, the bulk of the amplification product ranges in size from 200 to 400 base pairs. A size range of 150 to 250 base pairs is seen for damaged input RNA and picogram quantities of high quality RNA. Finally, integration of SeqPlex-amplified RNA with deep sequencing workflows requires the elimination of the primer sequences from each amplicon. An efficient primer removal step accomplishes this prior to sequencing sample preparation.
In addition to deep sequencing, SeqPlex-amplification product is suitable as microarray target or qPCR template, with or without primer removal.
SeqPlex RNA Amplification Kit Components
All components should be stored at –20 °C. When thawed for use, components should be kept on ice.
The reagents in this kit have been tested to assure that RNases are absent. The user, however, must protect the integrity of experimental results by wearing basic protective equipment, including gloved hands and lab coat. All reagent transfers throughout this procedure should be performed in a laminar flow hood or dedicated clean room. Frozen RNA samples should be thawed on ice.
Several components found in our SeqPlex RNA Amplification kit (SEQR), TransPlex® WTA kits, Complete Whole Transcriptome Amplification Kit (WTA2) and GenomePlex® WGA kits are similarly named. Though generally analogous in function, they are not interchangeable. Also, for users familiar with the WTA2 kit, please be aware that:
The SeqPlex RNA amplification kit provides for highly efficient removal of primer sequence following amplification. These primer sequences constitute 30 to 50% of amplification product mass and are removed by purification. Consequently, the quantity of amplification product used for input for the primer-removal reaction should be 2-fold of that required for sequencing.
The amount of nucleic acid required for sequencing is platform-specific, with Roche 454 minimally calling for ~1 µg of input nucleic acid. Illumina MiSeq and HiSeq platforms require as little as 50 ng, as does ABI SoLiD. Therefore, the procedure provided here is based on a final primer-removal reaction yield of 1 µg.
To be sure that you generate a sufficient quantity of amplified RNA, consult your sequencing service provider and scale the primer-removal reaction accordingly.
An amplification reaction will produce 2 -4 mg of amplified double-stranded cDNA when starting with 100 pg to 5 ng of high quality total RNA (RIN > 8.0). Higher input quantities and higher quality of RNA template generally result in increased yields. For damaged RNA, such as RNA isolated from FFPE (formalin-fixed paraffin embedded) samples, 1-50 ng input RNA is recommended. Reaction volumes can be scaled up or down to accommodate preparation of desired quantities of final product.
Genomic DNA must be removed from the RNA sample prior to amplification. If this was not accomplished during RNA isolation, use Product AMPD-1 RNase-free DNase following the kit instructions and using the most convenient library synthesis scale.
The SeqPlex RNA Amplification Kit will amplify ribosomal RNA, though less efficiently than messenger RNA. Ribosomal RNA depletion should be employed when required and feasible.
Library synthesis having a final recommended reaction volume of 5 µL is described below. However, a volume range of 2.5 through 25 µL (while maintaining an amplification reaction volume of 75 µL) has been shown to have no effect on amplification as detected by Ct values during early exponential amplification, amplification product yields, or β-actin transcript levels (see “Quality Control, Product Retention” below). Be sure to use the same library synthesis volume for all samples to be compared. Sufficient reagent has been supplied for the number of indicated kit reactions, at a library synthesis volume of 25 µL. Optional set-up instructions for increased reaction scale are provided following the recommended procedure.
rimer removal results in little or no loss of amplification product. However, a 75-µL primer- removal reaction input of 2 µg of amplified product is recommended to yield 1 µg of final product for entering the deep sequencing workflow. (See “Considerations for Downstream Sequencing” above.) This allows for loss due to primer removal plus any additional loss during reaction cleanup. A “no-enzyme” control reaction is also performed for each amplified sample. The control reaction is required to test for primer sequence removal (see Quality Control below).
Note: The amplification product cannot be further amplified after primer removal.
Note: Sufficient reagent has been included in the kit for a 75-µL “no-primer-removal-enzyme” control reaction. However, you may wish to reduce the scale of your “no enzyme control” to save reagent and amplified product, as shown below.
Note: A reaction setup described in step 15 is a mix that accounts for both the primer removal reaction and (steps 16 and 17) the no- enzyme control.
Amplification Product Size
Agilent Bioanalysis of amplified intact total RNA will show a smear, the bulk of which will range in size from 200 to 400 base pairs. Similar analysis of amplified damaged RNA or a low quantity input will range from 150 to 250 base pairs.
The efficiency of primer removal can be estimated by qPCR using the 5X Amplification Mix and Amplification Enzyme. Use unpurified primer removal reaction and corresponding control reaction for these assays (step 20). Sufficient amplification mix and enzyme are provided in the kit for a 15-µL qPCR test reaction for both the primer removal reaction and “no-primer-removal-enzyme” control reaction. A 1/1,000,000 dilution of each primer-removal and control reaction is used for this assay. Expect primer removal to be greater than 90%.
For the Primer Removal QC, combine reagents at following scale per single qPCR reaction:
3 µL 5X Amplification Mix (A5112)
0.15 µL Amplification Enzyme (5237)
1.85 µL 1/10,000 dilution, SYBR Green in 10 mM Tris-HCl, pH 8.0 (Product No. T3038)
10 µL 1/1,000,000 dilution cDNA (from primer removal reaction or no enzyme control)
94 °C, 2.5 minutes
94 °C for 30 seconds
70 °C for 5 minutes (read)
Expect a ΔCt of 3 – 7 as an estimate of successful primer removal:
(Ct)no primer removal enzyme – (Ct)plus primer removal enzyme
Note: Individual Primer Removal QC test reactions can be performed when necessary, as well as multiple reactions where a reagent mix is typically prepared (recommended). To pipet the small quantities indicated, use a pipette having the appropriate volume range and use siliconized or low-retention pipette tips. Prior to dispensing reagents, be sure any condensation that may have occurred during storage is spun down by centrifugation and appropriately mixed: by vortexing, pipeting or inversion.
Note: It has been demonstrated that the estimated percent primer removal determined with this assay correlates well with sequencing results.
Amplification Product Retention (Optional)
To demonstrate retention of amplification product during primer removal, an additional qPCR reaction(s) is performed using a primer pair(s) which encodes for a transcript(s) known to be expressed in the respective source sample. Include the no-enzyme control reaction in your experimental setup (see Primer Removal QC).
(For human RNA samples where b-actin is known to be expressed, the following primers can be used.)
Samples are now ready to enter the deep sequencing work flow. The bulk of SeqPlex amplification product ranges in size from 200 to 400 base pairs. Though typically unnecessary, additional fragmentation can be accomplished using sonication, a DNA sheering instrument (e.g., Covaris), or enzymatic fragmentation.
The terminus of each amplicon possesses a 5’-phosphate and 2-base 3’-over-hang. Prior to ligation of sequencing primers, polish double-stranded fragment ends with T4 polymerase. Ligation products comprising sequence chimeras have been reported for Roche 454 library preparation. 3’-adenylation in the Illumina workflow largely prevents this from occurring. A second sizing step prior to sequencing also helps to reduce the incidence of chimeras in the case of ABI SoLID sequencing.
The 5-µL volume scale for “Library Synthesis”, previously outlined, is recommended. For RNA sample volumes that cannot be adapted to this scale, the following reaction set-up instructions are provided.
Note: Input quantity is sometimes unknown, for example, when RNA is isolated from a small number of cells. Keep in mind that the number of amplification cycles required to reach plateau will vary as the quality and/or quantity of input RNA increases (fewer cycles) or decreases (more cycles).
The following table illustrates optional reaction volumes for Library Synthesis, step 2.
Table 2 proceeds with the reaction scale volumes established in Table 1, corresponding to Library Synthesis, step 4.
For Amplification, step 9, add the entirety of the library synthesis reaction to the 75-μL amplification reaction, adjusting water volume (Table 3).