Description
The SPLIT RNA Extraction Kit enables a fast and highly efficient extraction of RNA that is free of genomic DNA contamination. The RNA can be recovered as total RNA or split into two fractions, large RNA (> 150 nt) and small RNA (< 150 nt), facilitating the analysis of e.g. mRNA and miRNA from the same sample. Thus the RNA obtained is ideal for seamlessly preparing libraries for Next Generation Sequencing of total RNA or its large and small fractions.
With the content of the kit total RNA or the large RNA fraction can be extracted from 48 samples. If the small RNA fraction should be purified in addition to the large RNA fraction, then 24 samples can be extracted.
The SPLIT RNA Extraction Kit for Blood additionally includes the Blood Lysis Buffer for lysis of red blood cells prior to sample homogenization of the conventional SPLIT RNA Extraction Kit. This additional step enables efficient depletion of globin mRNA from the resulting RNA sample from as little as 50 to 250 µl of human blood.
Kit Content:
- Buffers and Purification Columns
- Phase-Lock Gel Columns
008 (SPLIT RNA Extraction Kit),
099 (SPLIT RNA Extraction Kit for Blood).
SPLIT RNA Extraction Kit
The SPLIT RNA Extraction Kit enables a fast and highly efficient extraction of RNA that is free of genomic DNA contamination. The RNA can be recovered as total RNA or split into two fractions, large RNA and small RNA, facilitating the analysis of e.g., mRNA and miRNA from the same sample. Thus the RNA obtained is ideal for seamlessly preparing libraries for Next Generation Sequencing of total RNA or its large and small fractions or any other demanding downstream application. The SPLIT RNA Extraction Kit for Blood enables concomitant depletion of globin mRNAs from human blood samples in low volumes (50 – 250 µl).
SPLIT Rapid Viral RNA/DNA Extraction Kit is available for fast and easy extraction of viral RNA from fluid samples (e.g. for SARS-CoV-2 analysis).
High Quality, High Yield
RNA extracted with the SPLIT RNA Extraction Kit 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.
Small RNA and Large RNA Fractions
The SPLIT kit can be used for the extraction of either total RNA (< 17 nt to > 10,000 nt) or for the isolation of the large RNA fraction (cut-off at ~ 150 nt), with the option to obtain the small RNA fraction separately (Figure 1).
Rapid Turnaround
Efficient miRNA Recovery
Efficient recovery of siRNA and miRNA down to 17 nt in the total RNA or in the small RNA fraction has been shown in spike-in experiments with small RNA markers (Figure 2).
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 from detectable genomic DNA contamination.
Free From Genomic DNA Contamination
Due to its highly optimized, phenol-extraction based protocol, the genomic DNA (gDNA) content in the extracted RNA sample is negligible compared to conventional methods (Figure 1).
No DNase Treatment, No RNA Degradation
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 RNA.
No gDNA Removal Column, No RNA Size Bias
The SPLIT workflow does not require the use of gDNA removal columns. Their function is based on size exclusion which can impose a size bias on the extracted RNA as well.
Figure 2. Separation of SPLIT RNA samples on a polyacrylamide gel, demonstrating the splitting of large and small RNA at a threshold of ~150 nt. The total RNA sample comprising small and large RNA is shown as comparison. The homogenate was spiked with a miRNA marker to assess efficient miRNA recovery.
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).
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 Lexogen’s QuantSeq 3’ mRNA-Seq (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 Kit for Blood. The number of discovered genes was calculated from CPM normalized read counts (threshold >0.5 CPM).
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Troubleshooting Guide
View the Troubleshooting Guide
Problem |
Likely cause |
Comments and suggestions |
No or poor phase separation |
Cooled phase-lock gel or insufficient centrifugation |
Ensure that phase-lock gel tubes are equilibrated at room temperature prior to the extractionEnsure that centrifugation is carried out at +18 °C or at room temperatureIncrease centrifugation time or repeat centrifugation step |
Incorrect cut-off |
Volume of isopropanol addition |
Measure volume of aqueous phase to add correct amount of isopropanol (0.33x , 1.75x, or 1x vol depending on the desired RNA fraction) |
Degraded RNA |
RNA source |
Avoid freeze-thaw cycles of your sampleFreeze starting material quickly in liquid nitrogen or in appropriate buffer |
RNase contamination |
Avoid speaking over opened tubes and wear glovesFollow protocol closely and work quickly |
|
Low absorption ratios (i.e., peak at 230 nm) |
Contamination with organic solvents, salts, or metal ions |
Ensure appropriate blank solution (e.g., Storage Buffer (SB) contains EDTA which can influence absorption measurements)Ensure to remove all residual GuSCN by following protocol closely (washing steps and dry spin)Optionally increase the number of washing steps |
gDNA contamination |
Contamination with organic phase |
Always decant upper phase and avoid disturbing of the organic phase via pipettingEnsure phase separation is complete by visual inspectionRepeat phenol-chloroform extractionDigest with RNase-free DNase according to manufacturer’s protocol but avoid heat inactivation of enzyme |
Low or no RNA yield |
RNA remains on spin column |
Increase incubation timePerform second elutionOptionally pre-heat Elution Buffer (EB) to 60 °C prior to elutionEnsure Wash Buffer (WB) has been diluted with 100 % ethanol as indicated on bottles |
Spin column was overloaded |
Reduce quantity of starting material (a maximum of 100 µg of RNA can be bound to the spin column) |
|
Problem in downstream application |
Salt or ethanol carry-over during elution |
Ensure Wash Buffer (WB) has been diluted with 100 % ethanol as indicated on bottlesIncrease number of washing stepsEnsure dry spin to remove all traces of ethanol |
Downloads
SPLIT RNA Extraction Kit
 User Guide – update 10.01.2020
 Application Note – SPLIT RNA Extraction Kit
Product Flyer – SPLIT RNA Extraction Kit for Blood
 SPLIT Supplementary Protocol: Purification of RNA from FFPE Samples – upload 18.07.2018
 008IM062V0110 – Instruction Manual for SPLIT Trial Kit – update 19.04.2016
 008MS024V0202 – MSDS Information for SPLIT RNA Extraction Kit – update 19.06.2020
Product Items
| Catalog Nr. | Product Name |
| 008.48 | SPLIT RNA Extraction Kit, 48 extractions |
| 099.48 | SPLIT RNA Extraction Kit for Blood, 48 extractions |
