RNA CleanUp and Concentration Kit

Supplier
Norgen Biotek
Cat. no.
1023600
Kit size
50
Links
Norgen Biotek Protocol

Features

• Efficient clean-up from various enzymatic reactions - RNA clean-up from upstream enzymatic applications such as labeling, DNase treatment and in vitro transcription. Remove proteins, RNases, DNases, nucleotides, etc.
• Clean-up and concentration of RNA isolated by different methods - RNA that has been isolated using various methods, including phenol-chloroform extractions or alcohol precipitations, can be processed with this kit.
• Fast and easy processing - Rapid spin-column format allows for the processing of 10 samples in 20 minutes.
• No phenol:chloroform extractions - Norgen

Norgen’s RNA Clean-Up and Concentration Micro Kit provides a rapid method for the purification, cleanup and concentration of up to 35 µg of RNA isolated using different methods including phenol/guanidine-based protocols, and from various upstream enzymatic reactions such as DNase treatment, labeling and in vitro transcription. The kit purifies all sizes of RNA, from large mRNA and ribosomal RNA down to microRNA (miRNA) and small interfering RNA (siRNA). The RNA is preferentially purified from other reaction components such as proteins, RNases and nucleotides, without the use of phenol or chloroform. The purified RNA is of the highest integrity, and can be used in a number of downstream applications including end-point or quantitative reverse transcription PCR, Northern blotting, RNase protection and primer extension, and expression array assays.

Purification is based on spin column chromatography using Norgen’s proprietary resin as the separation matrix. The RNA is preferentially purified from other cellular components such as proteins without the use of phenol or chloroform. The process involves first mixing the RNA samples or enzymatic reactions containing RNA with Binding Solution (please see the flow chart on page 4). Ethanol is then added and the mixture is loaded onto a spin-column. Norgen’s resin binds RNA in a manner that depends on ionic concentrations. Thus only the RNA will bind to the column, while the contaminating proteins or nucleotides will be removed in the flowthrough. The bound RNA is then washed three times with the provided Wash Solution in order to remove any remaining impurities, and the purified RNA is eluted with the Elution Buffer. The purified RNA is of the highest integrity, and can be used in a number of downstream applications. Norgen’s RNA Clean-Up and Concentration Micro Kit purifies RNA with minimal amounts of DNA contamination. An optional protocol is provided in Appendix A for maximum removal of residual DNA that may affect sensitive downstream applications such quantitative PCR.

Column Binding Capacity	35 µg
Size of RNA purified	All sizes, including small RNA (<200 nt)
Maximum Amount of Starting Material:	35 µg of RNA
Minimum Elution Volume	20 µL
Time to Complete 10 Purifications	20 minutes
Average Recovery	>90%

Concentration and Detection of Increasing Amounts of HeLa RNA

Increasing amounts of HeLa RNA in 50 µL input volumes were concentrated to 20 µL using Norgen’s RNA Clean-Up and Concentration Micro Kit. A 10 µL aliquot of the purified RNA was then used as the template in a qRT-PCR reaction to detect the S14 gene. The amounts indicated on the graph correspond to the amount of RNA that was used as the input for the qRT-PCR reaction, demonstrating the consistent performance of the kit even in the picogram range.

High Quality RNA

Total RNA was isolated from HeLa cells using TRI ® Reagent, which relies on organic extractions. The resulting RNA was then purified using Norgen’s RNA Clean-Up and Concentration Micro Kit, and a portion of the purified RNA was then run on an Agilent 2100 Bioanalyzer to demonstrate the high quality of the purified RNA.

User-Friendly Procedure

• Quantitative, real-time RT-PCR
• RT-PCR
• Northern blotting
• RNase protection and primer extension
• Expression array assays