KAPA RNA EvoPrep Kits

The poly(A) capture method used in an mRNA capture workflow is useful when specifically interrogating mRNA species. However, the workflow does bias towards exonic transcripts. The poly(A) capture approach also tends to have reduced 5' coverage of transcripts, due to the 3' polyadenylation of mRNA transcripts. 

The use of the KAPA RNA EvoPrep Kits with RiboErase (HMR) and/or RiboErase (HMR) Globin will allow a more accurate representation of the whole transcriptome and will contain all RNA species except rRNA and/or globin. This workflow retains intronic and intergenic regions, which is where many long non-coding transcripts are found.

Additionally, the poly(A) capture approach makes it incompatible for use with degraded RNA, where there is the possibility of strand breaks between the 3' polyadenylated region and the rest of the transcript. 

If you are using one of our KAPA RNA Hyper or Stranded RNA-Seq Kits, either with mRNA capture or rRNA and/or globin transcript depletion, please visit our online resource for Technical Documentation or alternatively, please Contact us. 

No, these kits are not compatible with small RNA. 

The KAPA RNA EvoPrep Kits (with no upfront enrichment) and the KAPA RNA EvoPrep Kits with RiboErase (HMR) and/or RiboErase (HMR) Globin are compatible with RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. The quality of FFPE-derived RNA can be highly variable due to the damaging nature of the formalin fixation process, where crosslinking, chemical modification, and fragmentation can occur. Library construction results may vary depending on the input amount and quality of the RNA. Higher RNA inputs may salvage library construction for particularly difficult FFPE samples. Please refer to the recommendations outlined in the Instructions for Use for each kit

KAPA mRNA EvoPrep Kits are only suitable for mRNA capture and library construction from high-quality input material (RIN ≥ 7). The use of fragmented RNA will result in strong bias towards the 3’-end of the mRNA.

The KAPA RNA EvoPrep Kits (with no upfront enrichment) are compatible with any species. The KAPA mRNA EvoPrep Kits are compatible with any species with 3’ poly-A tails. 

The KAPA RNA EvoPrep Kits with RiboErase (HMR) or RiboErase (HMR) Globin are compatible with human, mouse, and rat rRNA and/or globin transcript depletion.

Yes, both cytoplasmic and mitochondrial rRNA is depleted. 

Both the KAPA RNA EvoPrep Kit with RiboErase (HMR) and the KAPA RNA EvoPrep Kit with RiboErase (HMR) Globin use the same workflow and reagents, which allows for depletion of rRNA from human, mouse and rat species. The only difference is that an additional tube of depletion oligos are supplied and added during the initial hybridization step of the workflow, to deplete globin transcripts when using the KAPA RNA EvoPrep Kit with RiboErase (HMR) Globin. These globin depletion oligos target globin mRNA derived from hemoglobin alpha 1 (HGA1), hemoglobin alpha 2 (HGA2), hemoglobin beta (HGB), and hemoglobin gamma (HGG). 

• Fragmentation using heat and magnesium;  
• 1st strand cDNA synthesis using random priming; 
• Combined 2nd Strand cDNA Synthesis and A-tailing, which converts the cDNA:RNA hybrid to double-stranded cDNA (dscDNA), incorporates dUTP in the second cDNA strand and adds dAMP to the 3′-ends of the dscDNA library fragments;  
• Adapter ligation, where dsDNA adapters with 3′-dTMP overhangs are ligated to A-tailed library insert fragments; and  
• Library amplification to amplify library fragments carrying appropriate adapter sequences at both ends using high-fidelity, low-bias PCR. The strand marked with dUTP is not amplified, allowing strand specific sequencing. 

The upfront enrichment module for mRNA capture makes use of magnetic oligo-dT beads. The rRNA and/or globin depletion include hybridization of complementary DNA oligonucleotides, followed by treatment with RNase H and DNase to remove rRNA and/or globin transcripts duplexed to DNA and original DNA oligonucleotides, respectively.

KAPA HyperPure Beads are provided in this kit for reaction purification steps. It is a suspension of paramagnetic beads in a buffer optimized for purification in next-generation sequencing and other molecular biology workflows. KAPA HyperPure Beads are compatible with manual processing or automated liquid handling and enables efficient recovery in both formats. 

Yes, during 2nd strand synthesis, the cDNA:RNA hybrid is converted to dscDNA, with dUTP incorporated into the second cDNA strand. During library amplification, the strand containing dUTP is not amplified, allowing strand-specific sequencing. This kit retains accurate strand origin information in ˃99% of unique mapped reads. 

The sample preparation process may be paused safely after the upfront depletion modules as follows: 

After mRNA capture, the resuspended beads (in 22 μL of Fragment, Prime and Elute Buffer) may be stored at 4°C for ≤24 hours. 

After rRNA and/or globin depletion, following elution in 1X Fragment, Prime and Elute Buffer, samples may be stored at -20°C for ≤24 hours. 

The library construction process from RNA fragmentation through library amplification can be performed in approximately 3.5 hours, depending on the number of samples being processed, and experience. If necessary, the protocol may be paused safely after any of the following steps: 

• After the post-ligation cleanup, purified, adapter-ligated library may be stored at +2°C to +8°C for 1 – 2 weeks or at -15°C to -25°C for ≤1 month before amplification. 
• To avoid degradation, always store DNA in a buffered solution (10 mM Tris-HCl, pH 8.0 – 8.5) when possible, and minimize the number of freeze-thaw cycles.

RNA is fragmented using high temperature in the presence of magnesium. Depending on the origin and integrity of the input RNA, and the intended application, different RNA fragmentation protocols are provided to obtain the required insert size distribution. For intact RNA, such as that extracted from fresh/frozen tissue, longer fragmentation is required at higher temperatures. For degraded or fragmented RNA (e.g. from older samples or FFPE tissue), use a lower temperature and/or shorter times. The Instructions for Use for each product outlines various fragmentation parameters depending on the input RNA and the desired insert size. 

Compared with the KAPA RNA HyperPrep Kit and competitor kits, KAPA RNA EvoPrep offers enhanced ribosomal RNA depletion. This enables more consistent and reliable performance.¹

Ribodepletion is improved in the KAPA RNA EvoPrep Kit for a range of different inputs (5 ng, 25 ng and 100 ng input), and with low quality FFPET samples (our data shows reduced rRNA % at all three input levels for KAPA RNA EvoPrep Kit with RiboErase (HMR) compared with the KAPA RNA HyperPrep Kit with RiboErase (HMR) and four other supplier kits).¹

Compared with the KAPA RNA HyperPrep Kit and competitor kits, the KAPA RNA EvoPrep Kit provides enhanced strandedness to help produce reliable insights. This is across a range of inputs (5 ng, 25 ng and 100 ng), and with low quality FFPET samples.

KAPA Unique Dual-Indexed Adapters in both formats; truncated and full-length are recommended for use with the KAPA RNA EvoPrep Kits. However, these workflows are also compatible with other adapter designs wherein both the sequencing and cluster generation sequences are added during the ligation step, such as those routinely used in Illumina TruSeq, Agilent SureSelect XT2, and other similar library construction workflows. 

Custom adapters that are of similar design and are compatible with “TA-ligation” of dsDNA may also be used, remembering that custom adapter designs may impact library construction efficiency. Truncated adapter designs, where cluster generation sequences are added during amplification instead of ligation, may require modified post-ligation cleanup conditions. For assistance with adapter compatibility, please contact us. 

Please refer to the KAPA Adapters Webpage for more information and links to both the KAPA UDI Adapters and KAPA Hyperplex Adapters pages for more about barcode sequences, pooling, kit configurations, formulation, and for dilution for the different KAPA RNA EvoPrep Kits and inputs refer to the respective KAPA RNA EvoPrep Instructions for Use. 

Purified, adapter-ligated cDNA can be stored at 4°C for one week or at -20°C for at least one month, before amplification and/or sequencing. To avoid degradation, always store DNA in a buffered solution (10 mM Tris-HCl, pH 8.0) and minimize the number of freeze-thaw cycles. 

KAPA HiFi HotStart DNA Polymerase is the enzyme in the KAPA EvoAmp ReadyMix, provided in the KAPA RNA EvoPrep Kits. This is a novel B-family DNA polymerase engineered for low-bias, high fidelity PCR and is the reagent of choice for NGS library amplification.^1,2,3,4

1. Oyola, S.O. et al. BMC Genomics 13, 1 (2012).
2. Quail, M.A. et al. Nature Methods 9, 10–11 (2012).
3. Quail, M.A. et al. BMC Genomics 13, 341 (2012).
4. Ross, M.G. et al. Genome Biology 14, R51 (2013).

To minimize over-amplification and associated unwanted artefacts, the number of PCR cycles should be optimized to produce enough final, amplified library for downstream analysis and quality control, based on the Illumina instrument requirements. For target capture workflows, typically 1 µg of library yield is required, which may differ depending on the method used and the pre-capture multiplexing strategy employed. 

The number of cycles recommended in the Instructions for Uses for each KAPA RNA EvoPrep Kit should be used as a guide for library amplification, but cycle numbers may have to be adjusted depending on desired final library yield, library amplification efficiency, RNA fragmentation profile, and the presence of adapter dimers.

The size distribution of the dscDNA and/or final amplified library should be confirmed with an electrophoretic method. The quantification of the library should be performed with a qPCR based quantification kit such as the KAPA Library Quantification Kit for Illumina platforms. This kit employs primers based on the Illumina flow cell oligos, and can be used to quantify libraries that are ready for flow-cell amplification. 

The KAPA RNA EvoPrep Kits are supplied in multiple boxes. 

• The components for cDNA synthesis and library preparation are temperature sensitive, and should be stored at -15°C to -25°C in a constant-temperature freezer upon receipt. 
• The upfront enrichment module for mRNA capture, should be stored at 2°C to 8°C and for rRNA and/or globin depletion should be stored at -15°C to -25°C upon receipt.   
• Store KAPA HyperPure Beads at 2°C to 8°C. 

When stored under these conditions and handled correctly, the kit components will retain full activity until the expiry date indicated on the kit label.