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SPLIT RNA Extraction Kit

  • High-quality RNA for all downstream applications
  • Efficient extraction of total RNA, including small RNA
  • Universal – species-independent
  • Fast and convenient protocol – extracted RNA in 30 minutes


SPLIT RNA Extraction Kit

The SPLIT RNA Extraction Kit enables fast and highly efficient extraction of high-quality, high-purity RNA from various biological samples, including cell culture, animal and plant tissue, and fluid samples. The obtained RNA is ideal for seamless library preparation for Next Generation Sequencing and other demanding applications such as full-length reverse transcription, sample preparation for microarray analysis, or RT-qPCR. SPLIT recovers the complete RNA size range, including small RNAs (<200 nt). Additionally, large and small RNA-enriched fractions can be extracted by following a supplemental protocol.

High yields of high-quality RNA

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 (Figure 1). SPLIT RNA Extraction Kit extracts RNA with high efficiency. The typical yield of extracted RNA from mouse liver ranges from 4.0 – 4.5 µg total RNA / mg tissue.

Figure 1 | The SPLIT protocol enables the extraction of high-quality RNA. Gel-like representation of Agilent Bioanalyzer traces. RNA from mouse liver stored in RNAlater was extracted either as total RNA (lane 1) or as large and small RNA-enriched fractions (lanes 2 and 3). A control sample was extracted following a TRIzol protocol (lane 4). All samples have a RIN of 8.2 – 8.3 (not applicable for small RNA-enriched fraction).

Small and Large RNA-enriched 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-enriched fraction (cutoff at ~150 nt), with the option to obtain the small RNA-enriched fraction separately (Figure 1).

Universal, Species-independent RNA Extraction

SPLIT RNA Extraction Kit can be used with a broad range of material. A protocol is provided for animal and plant tissue, cell cultures, liquid samples, and FFPE samples.

Total RNA, Including miRNAs

SPLIT RNA Extraction Kit effectively extracts RNA of all size range, including small RNAs (≥17 nt). Efficient recovery of siRNAs and miRNAs down to 17 nt in total RNA or the small RNA-enriched fraction has been shown in spike-in experiments with small RNA markers (Figure 2).
Figure 2 | Separation of SPLIT RNA samples on a polyacrylamide gel, demonstrating the splitting of large and small RNA-enriched fractions at a threshold of ~150 nt. The total RNA sample comprising small and large RNA is shown as a comparison. The homogenate was spiked with a miRNA marker to assess efficiency of miRNA recovery. Small RNAs down to at least 17 nt are efficiently recovered in the total RNA sample and in the small RNA-enriched fraction.

Fast and Convenient Protocol

RNA can be extracted within 30 minutes. SPLIT RNA Extraction Kit contains Phase Lock Gel tubes allowing fast, comfortable, and safe phase separation.


The SPLIT RNA Extraction Kit contains reagents for the isolation of total RNA or the large RNA-enriched fraction from 48 samples, or small and large RNA-enriched fractions from 24 samples.

Total time:
30 min
The sample is homogenized in an isolation buffer that is highly chaotropic to facilitate effortless and complete solubilization.
The sample is transferred to a phase lock gel column which contains a special gel matrix that acts as a barrier between the organic and aqueous phase based on the density differences.
Acidic phenol and acidic buffer are added to create a monophasic solution, which is essential for the efficient separation of genomic DNA into the organic phase. Chloroform is added and phases are cleanly separated.
After centrifugation the gel acts as a seal between the phases.
The aqueous phase containing the RNA can be easily decanted and no carry-over of the organic phase will take place.
Depending on the amount of isopropanol added, either the total RNA (1.75x isopropanol) can be extracted, or the RNA can be split into a large (0.33x isopropanol) and a small RNA-enriched fraction (1x isopropanol to the flow-through of the large RNA-enriched fraction).
The RNA is precipitated onto a silica column. For the total RNA, the entire RNA will precipitate onto the silica carrier. For the large RNA-enriched fraction, RNA with a lower limit of about 150 nt will bind, whereas the small RNA will be in the flow-through.
The flow-through of the large RNA-enriched fraction contains the small RNAs which didn´t bind to the silica column. After the addition of 1x isopropanol, it can be precipitated and purified onto a new silica column.
10 – 50 µl of Elution Buffer or Storage Buffer are added to the silica membrane to elute the RNA bound to the silica carrier.

RNA extraction is finished and the RNA
is ready for anydownstream application.


Frequently Asked Questions

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Troubleshooting Guide

ProblemLikely causeComments and suggestions
No or poor phase separationCooled phase-lock gel or insufficient centrifugation
  • Ensure that phase-lock gel tubes are equilibrated at room temperature prior to the extraction
  • Ensure that centrifugation is carried out at +18 °C or at room temperature
  • Increase centrifugation time or repeat centrifugation step
Incorrect cut-offVolume 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 RNARNA source
  • Avoid freeze-thaw cycles of your sample
  • Freeze starting material quickly in liquid nitrogen or in appropriate buffer
RNase contamination
  • Avoid speaking over opened tubes and wear gloves
  • Follow 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
Low or no RNA yieldRNA remains on spin column
  • Increase incubation time
  • Perform second elution
  • Optionally pre-heat Elution Buffer (EB) to 60 °C prior to elution
  • Ensure 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 10 µg of RNA can be bound to the spin column)
Problem in downstream applicationSalt or ethanol carry-over during elution
  • Ensure Wash Buffer (WB) has been diluted with 100 % ethanol as indicated on bottles
  • Increase number of washing steps
  • Ensure dry spin to remove all traces of ethanol


SPLIT RNA Extraction Kit

Safety Data Sheet

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