Product 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 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.

Performance

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

RNA can be extracted within 30 minutes.

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

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

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.

Workflow

Step 1 : Cell/Tissue Homogenization

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.

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.

Step 2 : Phenol Extraction

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

Depending on the amount of isopropanol added either the total RNA (1.75x isopropanol) can be extracted or the RNA can be split in a large (0.33x isopropanol) and a small RNA fraction (1x isopropanol to the flow-through of the large RNA fraction).

Step 3 : Purification of RNA Fractions

Depending on the amount of isopropanol added either the total RNA (1.75x isopropanol) can be extracted or the RNA can be split in a large (0.33x isopropanol) and a small RNA fraction (1x isopropanol to the flow-through of the large RNA fraction).

The RNA is precipitated onto a silica column. For the total RNA the entire RNA will precipitate onto the silica carrier, while for the large fraction RNA with a lower limit of about 150 nt will bind
whereas the small RNA will be in the flow-through.

The RNA is precipitated onto a silica column. For the total RNA the entire RNA will precipitate onto the silica carrier, while for the large fraction RNA with a lower limit of about 150 nt will bind
whereas the small RNA will be in the flow-through.

Step 4 : Elution of RNA

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 any downstream application.

FAQs

Ordering Information

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User Guide – update 22.03.2016 (Workflow picture changed; Homogenization for plant tissue and fluid samples added; Condensation of column purification (total and small/large now one section); “Single” and “dual-fraction extraction” terms removed; List of species tested added.)
Application Note

Material Safety Datasheets

MSDS Information for SPLIT RNA Extraction Kit