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  • ‹ Back to RNA-seq
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Home › Learning centers › Next-generation sequencing › Technical notes › RNA-seq › Nonstranded libraries from FFPE inputs

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Tech Note

Prepare RNA-seq libraries from FFPE samples

  • rRNA removal and SMARTer cDNA synthesis: 
    Remove rRNA sequences and produce sequencing libraries from small amounts (10–100 ng) of degraded RNA (RIN 2–3)
  • RNA-seq data from FFPE samples: 
    Obtain complete transcriptome coverage from FFPE samples, using RiboGone - Mammalian and SMARTer random priming methods
Introduction Results Conclusions Methods

Introduction  

Next-generation sequencing (NGS) is a key tool for transcriptome analysis, with high sensitivity and a wide dynamic range. One challenge in NGS transcriptome analysis studies centers around FFPE (formaldehyde-fixed paraffin-embedded) tissue, in which the RNA is typically degraded.

Random-primed cDNA synthesis is an ideal solution for transcriptome analysis from FFPE tissue and other samples containing fragmented RNA; however, ribosomal RNA (which makes up ≥90% of total RNA) must be removed from these samples prior to cDNA synthesis. The RiboGone - Mammalian kit uses hybridization technology and RNase H digestion to identify and specifically degrade/eliminate 5S, 5.8S, 18S, and 28S nuclear rRNA sequences and 12S mitochondrial RNA sequences from RNA derived from human, mouse, or rat tissues.

Following RiboGone - Mammalian treatment, the RNA sample is ready for random-primed cDNA synthesis with the SMARTer Universal Low Input RNA Kit for Sequencing, which excels at cDNA amplification from low-input, fragmented RNA such as that found in FFPE tissue. The cDNA can then be prepared for sequencing using either the Illumina-specific ThruPLEX DNA-seq library preparation kit or Ion Torrent library preparation kits.

Results  

RNA extraction from FFPE tissue

Total RNA extracted from curls of breast carcinoma FFPE tissue (Cureline), using the NucleoSpin totalRNA FFPE kit according to its protocol, is degraded. The profile of the RNA is illustrated on an electropherogram trace with a broad peak at <200 bp.

Example electropherogram showing the degraded nature of FFPE RNA

Example electropherogram showing the degraded nature of FFPE RNA. Extracted RNA was validated on an Agilent 2100 Bioanalyzer with a RNA 6000 Pico Chip.

rRNA removal & random-primed SMARTer cDNA synthesis from low-input FFPE RNA

The RiboGone - Mammalian kit was used to clear rRNA from total RNA extracted from FFPE tissue. rRNA-depleted FFPE RNA was converted to cDNA with the SMARTer Universal Low Input RNA Kit for Sequencing according to the kit protocol. Illumina adapters and indices were added using the Low Input Library Prep Kit according to its protocol.

Workflow for random-primed cDNA synthesis with SMARTer universal low-input RNA kits.

Workflow for random-primed cDNA synthesis with SMARTer universal low-input RNA kits. SMARTer universal cDNA synthesis is random-primed, which makes it ideal for use with compromised mammalian RNA samples (e.g., RNA from FFPE tissue). Ribosomal RNA must be removed with RiboGone - Mammalian prior to SMARTer Universal cDNA synthesis.

High-quality RNA-seq data from FFPE samples

The library was sequenced on an Illumina MiSeq® instrument with ~6M 1 x 50 bp paired-end reads. rRNA reads were reduced to 0.6% of total reads, and 16,463 genes were identified. RiboGone treatment and random-primed SMARTer cDNA synthesis preserve transcriptome data while eliminating rRNA.

Sequencing data generated from FFPE samples

Sequencing data generated from FFPE samples. rRNA reads were reduced to 0.6% of total reads. The number of reads that mapped to introns, exons, intergenic regions, rRNA, mitochondrial RNA, and unknown sources are shown as percentages of the total reads.

Conclusions  

Random priming extends the applicability of transcriptome analysis to include samples which contain non-polyadenylated and/or compromised input RNA. However, in order to maximize RNA-seq data quality and quantity, random primed RNA-seq kits must be paired with rRNA removal methods. The RiboGone - Mammalian kit specifically removes 5S, 5.8S, 18S, and 28S rRNA sequences (as well as 12S mitochondrial rRNA sequences) from human, mouse, or rat total RNA. In this study, 16,463 genes were identified with an RPKM ≥0.1, while rRNA and mtRNA reads were reduced to <1% and ~2% of the RNA-seq library reads, respectively. These data indicate that SMARTer random-primed cDNA synthesis paired with RiboGone rRNA depletion yields high-value RNA-seq data, even from challenging samples such as small quantities of FFPE tissue.

Methods  

Total RNA was extracted from curls of breast carcinoma FFPE tissue (Cureline) using the NucleoSpin totalRNA FFPE kit according to its protocol, using lysis method B with a 75-minute incubation at 56°C and the optional on-column DNase treatment. 30 ng of the extracted total RNA was cleared of rRNA using the RiboGone - Mammalian kit according to the RiboGone kit protocol. 8 µl of rRNA-depleted FFPE RNA was converted to cDNA with the SMARTer Universal Low Input RNA Kit for Sequencing according to the kit protocol, using 18 PCR cycles for ds cDNA amplification due to the small amount and degraded nature of the RNA extracted from the FFPE curls. Illumina adapters and indices were added using the Low Input Library Prep Kit (now discontinued; replaced by the ThruPLEX DNA-Seq Kit) according to its protocol.

The RNA-seq library was sequenced on an Illumina MiSeq Platform with 1 x 50 bp reads. The reads were trimmed by CLC Genomics Workbench and mapped to rRNA, the mitochondrial genome, and the human genome with RefSeq masking using CLC (% reads indicated). 16,463 genes were identified with an RPKM (reads per kilobase of exon per million of reads) of at least 0.1. The number of reads that map to introns or exons is a percentage of the total reads.

Related products

Cat. # Product Size License Quantity Details
634938 SMARTer® Universal Low Input RNA Kit for Sequencing 10 Rxns USD $1104.00

The SMARTer Universal Low Input RNA Kit for Sequencing contains the components needed to synthesize high-quality cDNA from as little as 200 pg of RNA, and includes the Advantage 2 PCR Kit for PCR amplification and validation. The kit utilizes both SMART technology and random priming, and has been designed and validated to prepare cDNA samples for sequencing and quantification with next-generation sequencing platforms. While SMART technology offers unparalleled sensitivity and unbiased amplification of cDNA transcripts, random (universal) priming allows for amplification of damaged RNA and maintains the true representation of the original mRNA transcripts. Both of these factors are critical for transcriptome sequencing and gene expression analysis.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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634938: SMARTer Universal Low Input RNA Kit for Sequencing

634938: SMARTer Universal Low Input RNA Kit for Sequencing
634940 SMARTer® Universal Low Input RNA Kit for Sequencing 25 Rxns USD $2365.00

The SMARTer Universal Low Input RNA Kit for Sequencing contains the components needed to synthesize high-quality cDNA from as little as 200 pg of RNA, and includes the Advantage 2 PCR Kit for PCR amplification and validation. The kit utilizes both SMART technology and random priming, and has been designed and validated to prepare cDNA samples for sequencing and quantification with next-generation sequencing platforms. While SMART technology offers unparalleled sensitivity and unbiased amplification of cDNA transcripts, random (universal) priming allows for amplification of damaged RNA and maintains the true representation of the original mRNA transcripts. Both of these factors are critical for transcriptome sequencing and gene expression analysis.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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634846 RiboGone™ - Mammalian 6 Rxns USD $394.00

License Statement

ID Number  
246 This product is protected by U.S. Patent 9,428,794, and additional pending US and foreign patents. For further license information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.

RiboGone - Mammalian allows for the specific removal of rRNA sequences (5S, 5.8S, 18S, and 28S), as well as mitochondrial rRNA sequences (12S), from human, mouse, or rat total RNA. This kit is designed for use with limited sample amounts (10–100 ng of total RNA), and works with high- or low-quality RNA. Samples processed using the RiboGone kit are ready for cDNA synthesis with random primers.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Back

RiboGone - Mammalian removes rRNA efficiently from intact and degraded RNA, while retaining noncoding transcripts for analysis

RiboGone - Mammalian removes rRNA efficiently from intact and degraded RNA, while retaining noncoding transcripts for analysis
RiboGone - Mammalian removes rRNA efficiently from intact and degraded RNA, while retaining noncoding transcripts for analysis. RNA-seq libraries were generated from Human Brain Total RNA (Clontech) or Breast Cancer FFPE RNA (Cureline, extracted using a NucleoSpin totalRNA FFPE kit) and treated with the indicated rRNA removal method. Reads were mapped to the hg19 genome and read distributions were determined using Picard RNA-Seq Metrics. Libraries generated from RiboGone-treated RNA had comparably low rRNA reads to oligo(dT)-enriched RNA (Clontech), while retaining more noncoding reads.

Back

Sequencing data of RiboGone - Mammalian treated RNA samples highly correlate with MAQC qPCR data for the same reference RNA

Sequencing data of RiboGone - Mammalian treated RNA samples highly correlate with MAQC qPCR data for the same reference RNA
Sequencing data of RiboGone - Mammalian treated RNA samples highly correlate with MAQC qPCR data for the same reference RNA. The high level of correlation between the RNA-Seq and MAQC data (R=0.860) shows that depletion of rRNA with RiboGone does not interfere with detection of other genes by RNA-seq.

Back

Comparison of Human Universal Reference RNA and Human Brain Reference RNA sequencing alignment metrics for 10 ng of RNA treated with the RiboGone - Mammalian kit and the SMARTer Stranded RNA-Seq Kit show that RiboGone efficiently degrades rRNA from small total RNA samples

Comparison of Human Universal Reference RNA and Human Brain Reference RNA sequencing alignment metrics for 10 ng of RNA treated with the RiboGone - Mammalian kit and the SMARTer Stranded RNA-Seq Kit show that RiboGone efficiently degrades rRNA from small total RNA samples
Comparison of Human Universal Reference RNA and Human Brain Reference RNA sequencing alignment metrics for 10 ng of RNA treated with the RiboGone - Mammalian kit and the SMARTer Stranded RNA-Seq Kit show that RiboGone efficiently degrades rRNA from small total RNA samples.

Back

RiboGone - Mammalian is specifically designed to work with low input RNA samples containing between 10–100 ng of full-length or degraded total RNA

RiboGone - Mammalian is specifically designed to work with low input RNA samples containing between 10–100 ng of full-length or degraded total RNA
RiboGone - Mammalian is specifically designed to work with low input RNA samples containing between 10–100 ng of full-length or degraded total RNA. Samples processed using the RiboGone kit are ready for cDNA synthesis with any random-primed SMARTer RNA-Seq kit (including the SMARTer Stranded RNA-Seq Kit, the SMARTer Universal Low Input RNA Kit for Sequencing, and the SMARTer Universal Low Input RNA Library Prep Kit).

Back

634846: RiboGone - Mammalian

634846: RiboGone - Mammalian

Required Products

Cat. # Product Size Price License Quantity Details
634938 SMARTer® Universal Low Input RNA Kit for Sequencing 10 Rxns USD $1104.00

The SMARTer Universal Low Input RNA Kit for Sequencing contains the components needed to synthesize high-quality cDNA from as little as 200 pg of RNA, and includes the Advantage 2 PCR Kit for PCR amplification and validation. The kit utilizes both SMART technology and random priming, and has been designed and validated to prepare cDNA samples for sequencing and quantification with next-generation sequencing platforms. While SMART technology offers unparalleled sensitivity and unbiased amplification of cDNA transcripts, random (universal) priming allows for amplification of damaged RNA and maintains the true representation of the original mRNA transcripts. Both of these factors are critical for transcriptome sequencing and gene expression analysis.

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Back

634938: SMARTer Universal Low Input RNA Kit for Sequencing

634938: SMARTer Universal Low Input RNA Kit for Sequencing
634940 SMARTer® Universal Low Input RNA Kit for Sequencing 25 Rxns USD $2365.00

The SMARTer Universal Low Input RNA Kit for Sequencing contains the components needed to synthesize high-quality cDNA from as little as 200 pg of RNA, and includes the Advantage 2 PCR Kit for PCR amplification and validation. The kit utilizes both SMART technology and random priming, and has been designed and validated to prepare cDNA samples for sequencing and quantification with next-generation sequencing platforms. While SMART technology offers unparalleled sensitivity and unbiased amplification of cDNA transcripts, random (universal) priming allows for amplification of damaged RNA and maintains the true representation of the original mRNA transcripts. Both of these factors are critical for transcriptome sequencing and gene expression analysis.

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634940: SMARTer Universal Low Input RNA Kit for Sequencing

634940: SMARTer Universal Low Input RNA Kit for Sequencing
634847 RiboGone™ - Mammalian 24 Rxns USD $1377.00

License Statement

ID Number  
246 This product is protected by U.S. Patent 9,428,794, and additional pending US and foreign patents. For further license information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.

RiboGone - Mammalian allows for the specific removal of rRNA sequences (5S, 5.8S, 18S, and 28S), as well as mitochondrial rRNA sequences (12S), from human, mouse, or rat total RNA. This kit is designed for use with limited sample amounts (10–100 ng of total RNA), and works with high- or low-quality RNA. Samples processed using the RiboGone kit are ready for cDNA synthesis with random primers.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components You May Also Like Image Data Resources

Back

RiboGone - Mammalian removes rRNA efficiently from intact and degraded RNA, while retaining noncoding transcripts for analysis

RiboGone - Mammalian removes rRNA efficiently from intact and degraded RNA, while retaining noncoding transcripts for analysis
RiboGone - Mammalian removes rRNA efficiently from intact and degraded RNA, while retaining noncoding transcripts for analysis. RNA-seq libraries were generated from Human Brain Total RNA (Clontech) or Breast Cancer FFPE RNA (Cureline, extracted using a NucleoSpin totalRNA FFPE kit) and treated with the indicated rRNA removal method. Reads were mapped to the hg19 genome and read distributions were determined using Picard RNA-Seq Metrics. Libraries generated from RiboGone-treated RNA had comparably low rRNA reads to oligo(dT)-enriched RNA (Clontech), while retaining more noncoding reads.

Back

Sequencing data of RiboGone - Mammalian treated RNA samples highly correlate with MAQC qPCR data for the same reference RNA

Sequencing data of RiboGone - Mammalian treated RNA samples highly correlate with MAQC qPCR data for the same reference RNA
Sequencing data of RiboGone - Mammalian treated RNA samples highly correlate with MAQC qPCR data for the same reference RNA. The high level of correlation between the RNA-Seq and MAQC data (R=0.860) shows that depletion of rRNA with RiboGone does not interfere with detection of other genes by RNA-seq.

Back

Comparison of Human Universal Reference RNA and Human Brain Reference RNA sequencing alignment metrics for 10 ng of RNA treated with the RiboGone - Mammalian kit and the SMARTer Stranded RNA-Seq Kit show that RiboGone efficiently degrades rRNA from small total RNA samples

Comparison of Human Universal Reference RNA and Human Brain Reference RNA sequencing alignment metrics for 10 ng of RNA treated with the RiboGone - Mammalian kit and the SMARTer Stranded RNA-Seq Kit show that RiboGone efficiently degrades rRNA from small total RNA samples
Comparison of Human Universal Reference RNA and Human Brain Reference RNA sequencing alignment metrics for 10 ng of RNA treated with the RiboGone - Mammalian kit and the SMARTer Stranded RNA-Seq Kit show that RiboGone efficiently degrades rRNA from small total RNA samples.

Back

RiboGone - Mammalian is specifically designed to work with low input RNA samples containing between 10–100 ng of full-length or degraded total RNA

RiboGone - Mammalian is specifically designed to work with low input RNA samples containing between 10–100 ng of full-length or degraded total RNA
RiboGone - Mammalian is specifically designed to work with low input RNA samples containing between 10–100 ng of full-length or degraded total RNA. Samples processed using the RiboGone kit are ready for cDNA synthesis with any random-primed SMARTer RNA-Seq kit (including the SMARTer Stranded RNA-Seq Kit, the SMARTer Universal Low Input RNA Kit for Sequencing, and the SMARTer Universal Low Input RNA Library Prep Kit).

Back

634847: RiboGone - Mammalian

634847: RiboGone - Mammalian

Required Products

Cat. # Product Size Price License Quantity Details
634938 SMARTer® Universal Low Input RNA Kit for Sequencing 10 Rxns USD $1104.00

The SMARTer Universal Low Input RNA Kit for Sequencing contains the components needed to synthesize high-quality cDNA from as little as 200 pg of RNA, and includes the Advantage 2 PCR Kit for PCR amplification and validation. The kit utilizes both SMART technology and random priming, and has been designed and validated to prepare cDNA samples for sequencing and quantification with next-generation sequencing platforms. While SMART technology offers unparalleled sensitivity and unbiased amplification of cDNA transcripts, random (universal) priming allows for amplification of damaged RNA and maintains the true representation of the original mRNA transcripts. Both of these factors are critical for transcriptome sequencing and gene expression analysis.

Documents Components You May Also Like Image Data

Back

634938: SMARTer Universal Low Input RNA Kit for Sequencing

634938: SMARTer Universal Low Input RNA Kit for Sequencing
634940 SMARTer® Universal Low Input RNA Kit for Sequencing 25 Rxns USD $2365.00

The SMARTer Universal Low Input RNA Kit for Sequencing contains the components needed to synthesize high-quality cDNA from as little as 200 pg of RNA, and includes the Advantage 2 PCR Kit for PCR amplification and validation. The kit utilizes both SMART technology and random priming, and has been designed and validated to prepare cDNA samples for sequencing and quantification with next-generation sequencing platforms. While SMART technology offers unparalleled sensitivity and unbiased amplification of cDNA transcripts, random (universal) priming allows for amplification of damaged RNA and maintains the true representation of the original mRNA transcripts. Both of these factors are critical for transcriptome sequencing and gene expression analysis.

Documents Components You May Also Like Image Data

Back

634940: SMARTer Universal Low Input RNA Kit for Sequencing

634940: SMARTer Universal Low Input RNA Kit for Sequencing

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That's GOOD Science!

What does it take to generate good science? Careful planning, dedicated researchers, and the right tools. At Takara Bio, we thoughtfully develop best-in-class products to tackle your most challenging research problems, and have an expert team of technical support professionals to help you along the way, all at superior value.

Explore what makes good science possible

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Takara Bio USA, Inc. provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function. As a member of the Takara Bio Group, Takara Bio USA is part of a company that holds a leadership position in the global market and is committed to improving the human condition through biotechnology. Our mission is to develop high-quality innovative tools and services to accelerate discovery.

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES (EXCEPT AS SPECIFICALLY NOTED).

Clontech, TaKaRa, cellartis

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