SEQR - SeqPlex RNA Amplification Kit Protocol

Storage/Stability

All components should be stored at –20 °C. When thawed for use, components should be kept on ice.

RNA Handling Technique

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.

CAUTION:

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 Amplification Mix for the SeqPlex RNA Amplification kit is a 5X solution.
• The PCR amplification settings include final 30-minute 70 °C incubation.

Considerations for Downstream Sequencing

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.

Procedure

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.

Library Synthesis

1. Thaw the Library Synthesis Buffer, Library Synthesis Solution and water. Mix thoroughly by inversion or brief vortexing. Dissolve any precipitate in the Library Synthesis Solution (L8670) by briefly heating at 37 °C, followed by thorough mixing. Keep on ice.
   
   
2. Combine 100 pg to 5 ng of high quality intact total RNA or 1-50 ng damaged RNA (e.g., FFPE or laser capture sample RNA) with Library Synthesis Solution at the following single-reaction scale:
   
       0.5 µL Library Synthesis Solution (L8670)
       Add Nuclease-free water (W4502) to a total volume of 3.3 µL.
   
   
3. Mix by pipeting and incubate reactions in a thermocycler programmed for 70 °C for 5 minutes, then an 18 °C hold.  Do not hold at 18 °C for more than 10 minutes. Remove reactions from thermocycler, and place at room temperature or maintain at 18 °C for the next steps, but do not place on ice.
   
   Note: To avoid RNA renaturation and possible degradation, perform steps 4 and 5, and setup for step 6 as rapidly as possible at 18 °C or room temperature.
   
   
4. Prepare the following premix during the incubation in the previous step and immediately combine 1.7 µL of following premix with denatured RNA in Library Synthesis Solution (step 3):
   
       0.5 µL Library Synthesis Buffer (L9418)
       0.8 µL Water
       0.4 µL Library Synthesis Enzyme (L9543)
   
   
5. Mix by pipeting and spin down residue from the sides of reaction tubes.
   
   
6. Incubate in a thermocycler using the following conditions:
       18 ºC for 10 minutes
       25 ºC for 10 minutes
       37 ºC for 30 minutes
       42 ºC for 10 minutes
       70 ºC for 20 minutes
         4 ºC hold
   
   
7. Spin down any condensation by centrifugation. Samples may be amplified immediately or stored at –20 °C for up to one month.

Amplification

8. Thaw the 5X Amplification Mix. Dissolve any precipitate by briefly heating at 37 °C, followed by thorough mixing. Keep on ice.
   
   
9. Transfer the following reagents to the library synthesis reaction, using the following single-reaction scale*:
       53.50 µL Nuclease-free water
       15.00 µL 5X Amplification Mix (A5112)
       0.75 µL 1:1000 SYBR Green* in 10 mM Tris-HCl, pH 8.0 (Product No. T3038)
         0.75 µL Amplification Enzyme (A5237)
       75 µL Total reaction volume
   
   * Addition of SYBR Green, Product No. S9430, not included in the kit, is optional, but strongly recommended for monitoring amplification.  Prepare dilution and add immediately to the mix.  Discard dilution after each experiment.
   
   
10. Mix thoroughly by pipeting or brief vortexing. Spin down residue from top and sides of reaction tubes.
    
    
11. Proceed with PCR using the following thermocycler program (real-time qPCR is strongly recommended):
        1 cycle
            94 °C for 2 minutes.
        17-19 cycles*
            94 °C for 30 seconds
            70 °C for 5 minutes (read)
        1 cycle
            70 °C for 30 minutes
    
    Note: The final 70 °C incubation for 30 minutes is critical for primer removal and downstream sequencing application.
    
    * The optimal number of amplification cycles varies with RNA input quantity and quality. Optimal amplification is achieved by proceeding 2–3 cycles into the amplification “plateau”, as observed with real-time quantitative PCR. Typically, ~19 cycles are required for 1 ng to 5 ng of high quality RNA or 10 to 50 ng of FFPE RNA. In this case, if amplifying without real-time monitoring, performing multiple reactions, or otherwise unable to practically monitor individual amplification reactions, set the number of amplification cycles at 20. RNA of lower quality or quantity may require higher input quantities and/or more amplification cycles. If amplifying less than 1 ng of RNA (RIN > 8.0) or less than 10 ng of damaged RNA (RIN < 8.0) without real-time monitoring, performing multiple reactions, or otherwise unable to practically monitor individual amplification reactions, set the number of amplification cycles at 25. For best results, monitor amplification with Sybr Green.
    
    
12. After cycling is complete, maintain the reactions at 4 °C or store at –20 °C until ready for purification.
    
    
13. To remove residual primers and nucleotides, purify with the GenElute PCR Cleanup Kit (Product No. NA1020) as described in the kit instructions. Elute with 50 µL nuclease-free water, not kit elution buffer. Eluate can be concentrated by vacuum centrifugation if necessary, but avoid heating and do not allow the sample to go to dryness. (Because the amplification product is in water alone, without counter-ions present, the sample will denature upon dryness. Denaturation will inhibit primer removal.) The capacity of the GenElute purification column is 10 μg, adequate for purification of a typical amplification reaction.
    
    
14. Purified DNA is quantified by measuring absorbance. One A₂₆₀ unit is equivalent to 50 ng/µL DNA.

Primer Removal

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.

15. Combine and mix the following reagents:
    
        8.50 µL - 10x Primer Removal Buffer (SR401)
        1.70 µL - Primer Removal Solution (SR400)
        X      µL – 2.27 µg of purified SeqPlex-amplification product (step 14)
        Y      µL - Water (W4502)
    80.75 µL Total reaction volume
    
    
16. Transfer 9.5 µL of the mix in step 15 to a different reaction tube and add 0.5 µL water (W4502). This is the “no-enzyme” reaction. Mix thoroughly by pipeting. Spin down residue from top and sides of reaction tubes by brief centrifugation.
    
    
17. To the remaining 71.25 µL, add 3.75 µL Primer Removal Enzyme (SR402). This is the primer removal reaction. Mix thoroughly by pipeting. Spin down residue from top and sides of reaction tubes by brief centrifugation.
    
    
18. Incubate both primer removal and no-enzyme control reactions as follows:
        37 °C for 60 minutes
        65 °C for 20 minutes
          4  °C hold
    
    
19. Remove samples from the thermocycler and centrifuge briefly.
    
    
20. Reserve 2 µL each of the primer removal reaction, and the entire no-enzyme control reaction for the Quality Control assays below. Keep on ice or store at –20 °C for up to one month.
    
    
21. Purify the remaining primer removal reaction products using the GenElute PCR Clean-up Kit as described previously in step 13, or store unpurified samples at –20 °C for up to one month.
    
    
22. Primer removal reaction product yield and concentration is quantified by measuring absorbance. One A₂₆₀ unit is equivalent to 50 ng/µL DNA.

Quality 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.

Primer Removal

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)

    Amplification conditions:
        1 cycle
            94 °C, 2.5 minutes
        40 cycles
            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.)

    5’-CGGGACCTGACTGACTACCTC-3’
    5’-GAAGGAAGGCTGGAAGAGTGC-3’

1. Do not design primer pairs
   1. Where one of the primer pair aligns with any portion of the 3’-end of the upper strand of the double-stranded sequence shown immediately below.
      
                                                        Primer
                                             3’ ←——————— 5’
      5’-CTGAAGNNNNNNNNNNNNNNNN-3’
      3’-GACTTCNNNNNNNNNNNNNN      -5
      
      
   2. Or, where the primer pair amplifies a double-stranded DNA fragment that contains the following internal sequence:
      
                                                                                            Primer 1
                                                                                     3’ ←—————  5’
                                5’-CTGAAGNNNNNNNNNNNNNNNN-3’
                                3’-GACTTCNNNNNNNNNNNNNN      -5’
      5’ —————→ 3’
            Primer 2
   
   
2. Prepare 100 µM primer stock solutions.
   
   
3. Prepare a mix comprising a selected primer pair and 2X SYBR Green Jumpstart Taq ReadyMix (Product No. S4438).
   1. For 10 or more reactions:
      1. Determine the total quantity of 2X SYBR Green Jumpstart Taq ReadyMix required (10 µL per test reaction), equal to x.
      2. Divide x by 200, equal to y.
      3. Add y µL of each of the two primers directly to x µL 2X SYBR Green Jumpstart Taq and mix thoroughly by pipeting or vortexing.
      4. Combine
             10 µL 2X SYBR Green Jumpstart Taq ReadyMix + primers
             10 µL 1/10,000 dilution of primer-removal reaction or control
         
         
   2. Alternatively, for less than 10 reactions:
      1. Divide x by 20, equal to z.
      2. Add z µL of each of the two primers, diluted to 10 µM, directly to x µL 2X SYBR Green Jumpstart Taq and mix thoroughly by pipeting or vortexing.
      3. Setup qPCR reactions as follows:
             Combine
                11 µL 2X SYBR Green Jumpstart Taq ReadyMix + primers
                 9 µL 1/10,000 dilution of primer-removal reaction product or control
         
         
4. Perform qPCR using the following amplification conditions:
       1 cycle
           94 °C, 2.5 minutes
       40 cycles:
           94 °C, 30 seconds
           __°C, 30 seconds*
           72 °C, 30 seconds (read)
       Melt Curve, 55 °C to 95 °C, 1 °C per second, reading every second.
   
   * TM is primer specific.
   Expect to see the Ct value for your “+primer removal enzyme reaction” to be equivalent to your “–primer removal enzyme reaction” result, indicating full retention of amplification reaction product following primer-removal. However, expect to see a 30% to 50% mass loss by spectrophotometric detection due to primer removal.

Deep Sequencing

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.

Amplification Scale-Up (Optional)

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).