Catalog# : NB-21300
Price/Unit : $210.00
Unit : 25 preps
Rapid purification of microRNA without phenol; isolation and extraction of microRNA
* Specific enrichment of small RNAs without the use of phenol
* Rapid and conveneint spin-column protocol
* Process a wide range of samples
Bio-Synthesis's microRNA Purification Kit provides a rapid method for the isolation and purification of small RNA molecules from cultured animal cells, small tissue samples, bacterial cells, plants and blood in as little as 25 minutes. These small RNAs include regulatory RNA molecules such as microRNA (miRNA) and short interfering RNA (siRNA), as well as tRNA and 5S rRNA. Small RNA molecules are often studied due to their ability to regulate gene expression. miRNAs and siRNAs are typically 20-25 nucleotides long, and regulate gene expression by binding to mRNA molecules and affecting their stability or translation. Bio-Synthesis's microRNA Purification Kit purifies RNA molecules exclusively smaller than 200 nt, without the use of phenol or chloroform. microRNA could be isolated from a broad range of sample sources including cultured cells, small tissue samples, bacteria, yeast, fungi, plant and bodily fluids.
Purification is based on spin column chromatography using Bio-Synthesis's proprietary resin as the separation matrix. The process involves the use of two different spin columns: the Large RNA Removal Column and the microRNA Enrichment Column. Briefly, the cells or tissues of interest are lysed using the provided Lysis Buffer and ethanol is added to the sample. The lysate is then applied to the Large RNA Removal Column, and the larger RNA molecules will bind to Bio-Synthesis's resin while the smaller RNA species will pass through into the flowthrough (BIND 1).Ethanol is then added to the flowthrough, the sample is then applied to the microRNA Enrichment Column and the small RNA molecules will bind to Bio-Synthesis's resin (BIND 2). Only the RNA will bind to the column, and any remaining impurities are removed through a series of washes (WASH). The purified small RNA molecules are then eluted into 20 - 50 µL of the provided Elution Buffer or water (ELUTE). Please see procedure flowchart to the right.
Bio-Synthesis's microRNA Purification Kit preferentially purifies small RNA molecules from other cellular components such as ribosomal RNA without the use of phenol or chloroform. The purified RNA can be used in a number of downstream applications related to gene regulation and functional analysis, including northern blotting and microarray assays.
FEATURES AND BENEFITS
* Fast and easy processing - Rapid spin-column format allows for the processing of 10 samples in 30 minutes
* Streamlined protocol - Efficient isolation of small RNA species using a 2 column process, resulting in minimal contamination of larger RNA and genomic DNA
* No phenol:chloroform extractions - RNA is isolated without the use of harmful chemicals such as phenol or chloroform
* Specific small RNA purification - All small RNA species (<200>200 nt can also be easily purified using an optional protocol
APPLICATIONS
The purified RNA is of the highest quality and can be used in a number of downstream applications including:
* Bioanalyzer
* Quantitative, real-time RT-PCR for small RNA including miRNA
* RT-PCR for small RNA including miRNA
* Northern blotting
* RNase protection
* Primer extension
* Expression array assays
* Next Generation Sequencing
* microRNA Cloning
Figure 1. High Quality of Isolated RNA with Better Size Exclusion. Small RNA was isolated from HeLa cells using Bio-Synthesis's microRNA Purification Kit (Lanes C and D) and a competitor's kit (Lanes A and B). Ten microliters of the 50 µL purified small RNA were run on an 8% urea-agarose gel. Lane M is Bio-Synthesis's 100b RNA ladder. Note that Bio-Synthesis's kit is isolating only the small (<200>
Figure 2. Efficient Removal of Large RNA Species. Different RNA species were isolated from 106 HeLa cells, resolved on an Agilent® Lab-On-A-Chip and electropherograms were generated. Panel A contains all the RNA species present in 106 HeLa cells as isolated with Bio-Synthesis's Total RNA Purification Kit, and acts as a control. Panel B and C contain RNA that was isolated from 106 HeLa cells using Bio-Synthesis's microRNA Purification Kit. One microliter of the 50 µL purified RNA for each fraction was loaded. Panel B shows the large RNA species removed using the Large RNA Removal Columns, and no small RNA can be detected. Panel C shows the small RNA that is isolated using the microRNA Enrichment Columns, and shows that there is no contamination of the small RNA with any large RNA species above 200 nt. This demonstrates the effective separation of the small RNA from the large RNA species using Bio-Synthesis microRNA Purification Kit.
Figure 3. Effective Small RNA Amplification by End-Point RT-PCR. Bio-Synthesis's microRNA Purification Kit isolates small RNA that could be amplified in RT-PCR using the poly (A) polymerase extension method. Small RNA was isolated from 1 million HeLa cells, and 10 µL of the 20 µL isolated small RNA were polyadenylated in a 50 µL Poly-(A)-Polymerase reaction. Seven microliters of the polyadenylated RNA were used in a 20 µL reverse transcription reaction with a poly T adaptor primer. One microliter of the reverse transcription was used in a 20 µL PCR reaction with primers against the human microRNAs (miR-21) and 5S rRNA. Panel A shows the amplification of the miR-21 transcript from small RNAs while Panel B shows the 5S rRNA amplification from small RNAs. Lane 1 in both panels shows the results when total RNA isolated from 1 million HeLa cells using Bio-Synthesis's Total RNA Purification Kit was used as a control. Lanes 2 and 3 contain the successful RT-PCR when the microRNA isolated using Bio-Synthesis's microRNA Purification Kit was used as the template, and Lanes 4 contain the non-template control. The RT-PCR was successful for both reactions using the microRNA as the template. All PCR products were resolved on a 1X TAE, 3% agarose gel using Bio-Synthesis's PCRRanger as the molecular weight ladder.
microRNA Purification Kit Contents - Spin Columns
1. Lysis Solution
2. Wash Solution
3. Elution Buffer
4. Large RNA Removal Columns
5. microRNA Enrichment Columns
6. Collection Tubes
7. Elution Tubes
8 Product Insert
Long-Term Storage : All solutions should be kept tightly sealed and stored at room temperature. These reagents should remain stable for at least 1 year in their unopened containers.
Product Usage
For laboratory research use only, not for diagnostic use! Not for use in humans.
Reference/Citations :
Features
* Isolate all small RNAs
* Rapid protocol
* No phenol extractions
* Remove large RNAs
* Rapid spin-column format allows for the processing of 10 samples in 30 minutes.
* RNA is isolated without the use of harmful chemicals such as phenol or chloroform.
* All small RNA species can be isolated including miRNA, siRNA, tRNA and 5S rRNA.
* Efficient isolation of small RNA species using a 2 column process, resulting in minimal contamination of larger RNA and genomic DNA.
* Purified RNA can be used in a number of downstream applications relating to gene regulation and functional analysis, including northern blotting and microarray analysis.
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