Description

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 new SPLIT RNA Extraction Kit for Blood enables concomitant depletion of globin mRNAs from human blood samples in low volumes (50 – 250 µl).

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 (Figure 2).

RNA_Analysis_SPLIT_mRNA_Extraction_Kit

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.

NEW! 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).

SPLIT-Blood_Graphic

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).

Workflow

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

Cell/Tissue Homogenization

Step 1:

The sample is homogenized in an isolation buffer that is highly
chaotropic to facilitate effortless and complete solubilization.
The SPLIT for Blood protocol adds a red blood cell lysis step
prior to sample homogenization.

Cell/Tissue Homogenization

Step 1:

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.

Phenol Extraction

Step 2:

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.

Phenol Extraction

Step 2:

After centrifugation the gel acts as a seal between the phases.

Phenol Extraction

Step 2:

The aqueous phase containing the RNA can be easily decanted
and no carry-over of the organic phase will take place.

Purification of RNA Fractions

Step 3:

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).

Purification of RNA Fractions

Step 3:

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.

Purification of RNA Fractions

Step 3:

The flow-through of the large RNA fraction contains the small RNAs
which didn´t bind to the silica column. After addition of 1x isopropanol
it can be precipitated and purified onto a new silica column.

Elution of RNA

Step 4:

10 – 50 µl of Elution Buffer or Storage Buffer are added to the silica
membrane to elute the RNA bound to the silica carrier.

Elution of RNA

Step 4:

RNA extraction is finished and the RNA is ready for any
downstream application.

For viewing the whole workflow on page please click here

Featured Publications

List of the most recent SPLIT publications.

FAQ

Frequently Asked Questions

Please find a list of the most frequently asked questions below. If you cannot find the answer to your question here or want to know more about our products, please contact support@lexogen.com.

1. How is genomic DNA removed with the SPLIT RNA Extraction Kit?

The SPLIT protocol efficiently removes genomic DNA from most cell types with the help of acidic phenol. However, certain sample types, such as blood, contain unusually high proportions of DNA in comparison to RNA. Due to this imbalance, gDNA carry over may occur. This can be seen as a well-retarded band in agarose gel runs and on microfluidic devises such as the Fragment Analyzer in setups that allow for simultaneous analysis of RNA and high-molecular weight DNA. For these difficult sample types, DNase treatment may be required prior to processing of the extracted RNA. For more information please contact us (support@lexogen.com), we gladly support you with protocol suggestions tailored to your workflow.

2. How long does the protocol take?

The extraction of the large RNA fraction or the total RNA takes around 30 min. If small and large RNAs are to be extracted 35 min can be estimated. The whole workflow can be seen in the Schematic overview of the SPLIT workflow, SPLIT User Guide (page 5).

3. Which RNA types can I expect in the small and large RNA fractions?

The SPLIT protocol provides you with a size cut off at ~150 nt. The small RNA fraction (< 150 nt) will contain miRNA, tRNA, 5 S rRNA, and 5.8 S rRNA, while the large RNA fraction (>150 nt) will contain mRNA, 18 S rRNA, 28 S rRNA, and snRNA.

4. What is the typical yield of RNA to be extracted with the protocol?

The average yield of RNA obtained will depend on the sample type being used, the amount of starting material, the species, growth conditions, and the developmental stage of the sample. For instance a typical RNA extraction from mouse liver yields approximately 4.0 – 4.5 µg total RNA/mg tissue, of which 3.0 – 3.5 µg contribute to the large RNA fraction and 0.6 µg originate from small RNAs. The general yield for mouse brain is 0.65 µg for large and 0.3 µg small RNAs/mg tissue and for 1 x 10⁶ human cells 10.0 – 11.5 µg for the large and 1.0 – 1.1 µg for the small RNA fraction.

5. For which organisms does the SPLIT Kit work?

SPLIT has been tested for isolation of RNA from different organisms including animal (e.g., mouse, human) and plant tissues (e.g., A. thaliana, Picea abies), insects (e.g., drosophila), cell lines (e.g., human), fluid samples (e.g., plasma), and others (jellyfish, fungi, bacteria). For more information please contact us (support@lexogen.com), we will gladly support you with protocol adaptations.

The SPLIT RNA Extraction Kit for Blood protocol is verified for use with 50 – 250 µl of fresh human blood collected in EDTA containing blood collection tubes. Please contact support@lexogen.com if you wish to use this protocol with blood from other species, variable input blood volumes, or with blood exposed to other collection methods or storage conditions.

6. Do I need to purchase any extra reagents to perform the extraction?

Acidic phenol, chloroform, ethanol and isopropanol have to be supplied by the user. We recommend purchasing the phenol solution pH 4.3 at Sigma-Aldrich (P4682-100ML). For the complete list User-supplied Consumables and Equipment, SPLIT User Guide (page 7)

7. Is the RNA extracted with the SPLIT kit only appropriate for RNA-Seq?

RNA-Seq is a demanding application, performing best with high quality RNA, but high-quality RNA extracted with the SPLIT protocol is suitable for any other downstream application as well.

8. How many RNA extractions can be done with the SPLIT Kit?

Either 24 reactions isolating large RNAs plus 24 reactions isolating small RNAs or alternatively 48 reactions isolating only the large RNAs or total RNA can be performed.

9. What is the minimum input amount?

High quality RNA was successfully extracted with the SPLIT RNA Extraction Kit using 0.5 mg of tissue or 100 cells. Using the SPLIT RNA Extraction Kit for Blood protocol high quality RNA can be extracted from 50 – 250 µl of fresh human blood collected in EDTA containing blood collection tubes. Using 50 µl of whole blood RNA is the maximum volume we could recommend when BLB is not used for SPLIT RNA Extraction (i.e. extracting RNA from whole blood).

10. Can the SPLIT kit be used to extract RNA from plasma samples?

Yes, the SPLIT RNA Extraction Kit also works for the extraction of total RNA or the small and large RNA fractions from plasma. For protocol specifications see SPLIT User Guide, 5.1.4. Fluid samples (p. 13, step 1).

11. Can I use SPLIT to separate small and large RNA fractions from already extracted RNA?

Yes, this is possible. For this, add 400 µl IB and 150 µl AB to a small volume of your total RNA (e.g. up to 100 µl). Mix properly and add 0.33x volume of isopropanol to this mixture, vortex for 10 seconds and continue at SPLIT User Guide, 5.3.1.2. Column Loading of Large RNA (p. 17, step 18).

12. Can I extract DNA with the SPLIT kit?

No, the kit is for RNA extraction only. However, since SPLIT is based on acidic phenol-chloroform extraction, DNA and proteins will be located in the lower organic phase. Therefore, you simply have to recover and purify the organic phase. For more information please contact us (support@lexogen.com), we gladly support you with protocol suggestions.

13. What is the maximum input amount for the SPLIT kit?

The maximum binding capacity of the purification column is 100 µg RNA, which should not be exceeded for optimal results. For mouse liver tissue this translates into an upper limit of 20 – 25 mg input per extraction for example.

14. Do you have SPLIT RNA Extraction Kit for Blood also available for other species than human?

The protocol is verified for use with 50 – 250 µl of fresh human blood collected in EDTA containing blood collection tubes. Please contact support@lexogen.com if you wish to use this protocol with blood from other species, variable input blood volumes, or with blood exposed to other collection methods or storage conditions.

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 extraction Ensure that centrifugation is carried out at +18 °C or at room temperature Increase 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 sample Freeze starting material quickly in liquid nitrogen or in appropriate buffer
Degraded RNA	RNase contamination	Avoid speaking over opened tubes and wear gloves Follow protocol closely and work quickly
Low adsorption 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 adsorption 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 pipetting Ensure phase separation is complete by visual inspection Repeat phenol-chloroform extraction Digest 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 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
Low or no RNA yield	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 bottles Increase number of washing steps Ensure dry spin to remove all traces of ethanol

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