RNA Extraction and cDNA amplification - Lexogen

Reagents & Kits

RNA Extraction and cDNA amplification - Lexogen 

In NGS experiments, the library prep is a very important aspect of the entire workflow, as the quality of library prep will greatly influence the quality of the sequencing data.

But, next to the Library Prep, also the quality of the input material is crucial. In order to maximize the success ratio of your NGS experiments, we do offer 2 kits for RNA extraction and long-read cDNA amplification that result in high quality material to use in downstream Library Prep.

•    SPLIT RNA Extraction Kit
•    TeloPrime Full Length cDNA Amplification Kit v2 

•    SPLIT RNA Extraction Kit

The SPLIT RNA Extraction kit offers fast and efficient extraction of RNA, free from Genomic DNA. The RNA can be recovered as total RNA or split into 2 fractions, large RNA and small RNA, enabling the efficient analysis of e.g. mRNA and miRNA from the same sample. 

The obtained RNA is ideal for seamlessly preparing NGS libraries of total RNA or its large and small fractions or any other demanding downstream application.
The dedicated SPLIT kit for Blood enables concomitant depletion of globin mRNAs from human blood samples in low volumes (50-250µl).

o    High quality RNA for demanding downstream applications
o    Total RNA or small and large RNA fractions
o    No Dnase treatment; no RNA degradation
o    Also available for RNA extraction from blood
o    Efficient depletion of globin mRNA
o    Rapid turnaround ( 30 minutes)

High Quality, High Yield

The extracted RNA has a high RIN quality score for all types of samples. A RIN of 10 and a 28S/18S rRNA ratio of 2.7 can be obtained from cell culture. Extractions from tissue samples usually result in RNA with a RIN of 8.0-9.5.

Efficient miRNA recovery
 

SPLIT RNA extraction enables efficient recovery of siRNA and miRNA down to 17nt in the total RNA or in the small RNA fraction. This has been shown in spike-in experiments with small RNA markers.

Free from Genomic DNA contamination

The highly optimized, phenol-extraction based protocol ensures negligible gDNA content in the extracted RNA sample, compared to conventional methods.

figure 1: Agarose gel analysis of RNA samples extracted with the SPLIT kit or by a TRIzol/isopropanol precipitation method. In the TRIzol extracted sample genomic DNA is visible as a slot-retained band, whereas RNA obtained with the SPLIT kit is free of detectable genomic DNA contamination

Small and Large RNA fractions
 
The SPLIT kit can be used for the extraction of either total RNA ( 17nt to  10.000nt) or for the isolation of the large RNA fraction (cut-off at ±150nt), with the option to obtain the small RNA fraction separately

figure 2: Separation of SPLIT RNA samples on a PA-gel, demonstrating the splitting of large and small RNA at a threshold of 150nt. The total RNA sample comprising small and large RNA is shown as a comparison. The homogenate was spiked with a miRNA marker to assess efficient miRNA recovery

No DNase treatment, No RNA degradation, No gDNA removal column, No RNA size Bias

The SPLIT protocol does not require DNase-treatment which is often used for the removal of genomic DNA in the sample and can be a reason for degradation of the RNA.
In addition, the SPLIT workflow does not use gDNA removal columns either. The function of those columns is based on size exclusion, which can impose a size bias on the extracted RNA as well.

RNA extraction from Blood
The SPLIT RNA extraction kit for Blood includes an additional step which enables efficient depletion of predominant globin mRNA from human blood samples. This results in increased Gene Detection.

figure 3: A: The SPLIT for blood protocol depletes >95% of globin mRNA species from the extracted RNA samples. RNA was extracted from fresh human blood using the SPLIT (left side bar) and SPLIT for Blood protocol (right side bar), respectively. RNA-seq libraries were prepared with QuantSeq 3’ mRNA FWD kit and sequenced reads were mapped to the human reference genome and the percentage of reads mapping to globins was calculated.
B: Increased gene detection in human blood QuantSeq libraries using SPLIT RNA extraction for Blood. The number of discovered genes was calculated from CPM normalized read counts (threshold >0.5 CPM)

Ordering information: 

Contact us for pricing and additional information 

Cat. n°

Description

Reaction size

008.48

SPLIT RNA Extraction Kit

48 preps

099.48

SPLIT RNA Extraction Kit for Blood

48 preps

•    TeloPrime Full-Length cDNA Amplification Kit v2

The TeloPrime Full-Length cDNA Amplification Kit V2 is an all-in-one protocol for generating full-length DNA from total RNA, based on Lexogen unique Cap-Dependent Linker Ligation (CDLL) and long reverse transcription (long RT) technology, and is highly selective for full-length RNA molecules that are both capped and polyadenylated.

o    Ideal for long-read NGS library Preparation for Pac-Bio or Oxford Nanopore sequencing
o    Exceptional 5’ cap specificity
o    Full length cDNA generation with high yield
o    Flexible input requirements (1ng-2µg total RNA)
o    Flexible protocol enabling use of custom primers for Reverse Transcription

Exceptional Elimination of cDNA from Degraded RNA
 
The ligation of the 5’ linker to the end of the 3’ end of the cDNA is based on the Cap-Dependent Linker Ligation (CDLL) technology and happens in a highly cap-dependent manner. This provides an exceptional cap-specificity and eliminates cDNA products from degraded mRNA.

Full-Length cDNA synthesis

cDNA synthesis is based on the unique CDLL and Long reverse transcription (Long RT) technology from Lexogen. This makes TeloPrime highly selective for full-length RNA molecules that are both capped and polyadenylated.

Multiple Downstream Applications

The via TeloPrime generated full-length cDNAs can be used for a great variety of downstream applications (eg. NGS, RACE, cloning, microarray probes and normalization). This kit enables detection and correct quantifications of splice variants and their true transcription start- and end-sites, in both short and long mRNA molecules. For further full-length or gene-specific PCR’s, a TeloPrime PCR Add-on Kit v2 is offered, containing the PCR forward and reverse primer separately, enabling substitution with the gene-specific primer of interest.
 

figure 4: In an mRNA-seq experiment, mRNAs were tagged at their 5' end using TeloPrime, Template-Switch (TS) or oligo capping (OC). Transcript start sites (TSS) were mapped to the mouse genome. The accumulated TSS read coverage is plotted against the normalized annotated transcript length to show relative TSS mapping for the top 500 expressed genes.

Ordering information

Contact us for pricing and additional information 
 

Cat. n°

Description

Reaction size

013.08

TeloPrime Full-Length CDNA Amplification Kit V2

8 preps

013.24

TeloPrime Full-Length CDNA Amplification Kit V2

24 preps

018.16

TeloPrime PCR Add-on Kit V2

16 rxn

 

LEARN MORE ABOUT RNA EXTRACTION AND cDNA AMPLIFICATIONExtraction and cDNA amplification

Request a quote or ask your question about RNA Extraction and cDNA amplification via the contact options below. We will try to answer your question within one working day.

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+31 30 6880771
info@isogen-lifescience.com

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