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Comprehensive comparative analysis of RNA sequencing methods for degraded or low input samples

ABSTRACT: RNA-Seq is an effective method to study the transcriptome, but can be difficult to apply to scarce or degraded RNA from fixed clinical samples, rare cell populations, or cadavers. Recent studies have proposed several methods for RNA-Seq of low quality and/or low quantity samples, but their relative merits have not been systematically analyzed. Here, we compare five such methods using a comprehensive set of metrics, relevant to applications such as transcriptome annotation, transcript discovery, and gene expression. Using a single human RNA sample, we constructed and deeply sequenced 10 libraries with these methods and two control libraries. We find that the RNase H method performed best for low quality RNA, and can even effectively replace oligo (dT) based methods for standard RNA-Seq. SMART and NuGEN had distinct strengths for low quantity RNA. Our analysis allows biologists to select the most suitable methods and provides a benchmark for future method development.

ORGANISM(S): Homo sapiens

PROVIDER: GSE40705 | GEO | 2013/05/15

SECONDARY ACCESSION(S): PRJNA174728

REPOSITORIES: GEO

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Project description:Purpose: The goals of this study is to determine the best method of gene expression quantification (RNA-seq, Microarray, NanoString) and amplification kits adapted to low-input and/or low-quality RNA samples (FFPE samples) Methods: Mouse bladder cancer cell line (mouse bladder cancer cell line, BC57) and mouse normal mouse normal urothelium were fixed in formalin and embedded in paraffin (FFPE), andfesh frozen (FF) in liquid nitrogen. The total RNA of these 4 samples were tested by 3 technologies (NanoString, RNA-seq and Microarray) and the results were compared to its reference (high-quality and high-input RNA of mouse bladder cancer cell line and mouse normal mouse normal urothelium). For NanoString with low-input RNA samples, each sample was tested by NanoString quantification after amplification by SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian and Ovation SoLo NuGEN RNA-seq System, and NanoString based on PCR approach with three input quantities: 50pg, 250pg and 2ng of total RNA, except for NanoString quantification after amplification by SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian kit for which the minimum recommended quantity was 250pg of total RNA. NanoString direct quantification was also done for FF and FFPE samples at high amount (50ng of total RNA) and results obtained from FF samples were considered as the reference. To determine which is the method for NanoString technology, low-input and low-quality RNA samples, we performed NanoString control quality metrics, principal component analysis, and a differential analysis between the mouse bladder cancer cell lines and the mouse normal mouse normal urothelium for each input quantity, amplification method and method of sample preservation (FF or FFPE). Results: The NanoString based PCR based approach is recommended for quantification of gene expression of FFPE and FF samples from 250pg of total RNA. However, NanoString quantification after amplification by SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian and Ovation SoLo NuGEN RNA-seq System is not recommended for FF and FFPE from low-input samples.

Project description:Purpose: The goals of this study is to determine the best method of gene expression quantification (RNA-seq, Microarray, NanoString) and amplification kits adapted to low-input and/or low-quality RNA samples (FFPE samples) Methods: Mouse bladder cancer cell line (mouse bladder cancer cell line, BC57) and mouse normal mouse normal urothelium were fixed in formalin and embedded in paraffin (FFPE), andfesh frozen (FF) in liquid nitrogen. The total RNA of these 4 samples were tested in triplicate by 3 technologies (NanoString, RNA-seq and Microarray) and the results were compared to its reference (high-quality and high-input RNA of mouse bladder cancer cell line and mouse normal mouse normal urothelium). For RNA-sequencing, each sample was tested by two library contruction kits: SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian and Ovation SoLo NuGEN RNA-seq System with three input quantities: 50pg, 250pg and 2ng of total RNA, except for the SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian kit for which the minimum recommended quantity was 250pg of total RNA. RNA-seq based on poly(A) tail enrichment was considered as the reference and was done for the two FF samples at high amount (1µg of total RNA) . Results obtained with the two tested kits were compared to those based on poly(A) tail enrichment. To determine which is the best kit suitable for RNA-seq, low-input and low-quality RNA samples, we performed RNA-seq control quality metrics, principal component analysis, and a differential analysis between the mouse bladder cancer cell lines and the mouse normal mouse normal urothelium for each input quantity, library kit and method of sample preservation (FF or FFPE). Results: The Ovation SoLo NuGEN RNA-seq System library kit are recommended for quantification of gene expression of FFPE and FF samples at low-input (down to 50pg) whereas the SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian is only suitable for FF samples from 250pg of total RNA.

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Comprehensive comparative analysis of RNA sequencing methods for degraded or low input samples

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