Catalog

17200

Total RNA Purification Kit

( 50 preps )
Norgen Biotek

443.76$

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Additional description

Total RNA Purification Kit (Cat# 17200 )

For rapid purification of total RNA - including microRNA - without phenol

  • IsolatetruetotalRNA including siRNA and microRNA
  • No phenol extractions
  • Purify high-quality RNA in 20 minutes
  • Extract RNA from as little as a single cell
  • Isolate from a wide variety of specimens

Product Description

Norgen’s Total RNA Purification Kit provides a rapid method for the isolation and purification of total RNA in as little as 20 minutes. The kit purifies all sizes of RNA, from large mRNA and ribosomal RNA down to microRNA (miRNA) and small interfering RNA (siRNA), without the use of phenol or chloroform.Norgen's kitis the only kiton the market that isolates true total RNA, as other kits must use phenol to recover all sizes of RNA. Therefore Norgen's kitoffers significant advantages in functionality,savings on cost,ease-of-use,no hazardous organic waste, and noinhibitory effect onPCR amplifications as a result of residual phenol. With this kit both miRNA and mRNA can be studied from the same sample without further purifications, thusoffering considerable advantages when comparing and relating expression of miRNA to other RNA. Furthermore, this is an excellent kit for the extraction of miRNA from all samples including plasma.

Total RNA can be isolated from a broad range of sample sources with this kit includingcultured cells, tissues, blood, serum, plasma, bacteria, yeast, fungi, plants and viruses.Norgen's Total Purification RNA Kit works very well with small sample inputs and does not require carrier RNA for the extraction. Total RNA can be isolated from as little as a single cell using this kit.

Purification is based on spin column chromatography using Norgen’s proprietary resin as the separation matrix. Briefly, the cells or tissue of interestare first lysed using the provided Lysis Solution, ethanolis addedand the RNA isbound to Norgen's column (BIND). Under these conditions only the RNA will bind to Norgen's resin while most of the contaminating cellular proteins areremoved in the flowthrough or retained on top of the resin. The boundRNAis then washed to remove any remaining impurities(WASH). Lastly, the purified total RNA is eluted into 50 µL of the provided Elution Buffer or water (ELUTE). Please see procedure flowchart to the right.
Norgen’s proprietary resin provides superior affinity to the full size range of RNA molecules, resulting in large and small RNA (miRNA) purification with better linearity and sensitivity. The purified RNA is of the highest integrity, and can be used in a number of downstream applications including real time RT-PCR,RT-PCR, Northern blotting, RNase protection and primer extension, expression profiling, miRNA cloning and amplificationand Next Generation Sequencing.

FEATURES AND BENEFITS

  • No phenol:chloroform extractions - Total RNA is isolated without the use of harmful chemicals such as phenol or chloroform
  • Isolate total RNA from very small samples - Total RNA has been isolated and detected from as little as a single animal cell
  • Extremely efficient isolation of low abundance microRNA - Norgen's Total RNA Purification Kit has been shown to be extremely efficient at recovering low abundance microRNA from plasma samples (link)
  • Isolate a diversity of RNA species - All RNA species can be isolated, from large mRNA and ribosomal RNA down to microRNA (miRNA) and small interfering RNA (siRNA)
  • Fast and easy processing - Rapid spin-column format allows for the processing of 10 samples in 20 minutes
  • Isolate total RNA from a broad input source - Total RNA has been isolated from cultured animal cells, small tissue samples,LCM samples, bacteria, yeast, fungi, plants, viruses and various bodily fluid including blood, plasma, serum, saliva, nasal or throat swabs
  • No need for carrier RNA - Isolate all sizes of RNA without the use of carrier RNA
  • Multiple kit sizes available - This kit is available in both 50 prep and 100 prep sizes

Specifications

  • Quantitative, real-time RT-PCR for both large mRNA and small RNA including miRNA
  • RT-PCR for both large mRNA and small RNA including miRNA
  • Expression profiling
  • Next Generation Sequencing for RNA and miRNA
  • miRNAfrom plasma for discovery
  • microRNA cloning and amplification
  • PCR-based virus detection
  • PCR-based viable bacteria detection
  • Northern blotting
  • RNase protection
  • Primer extension

Figure 1. High Quality of Isolated RNA with Complete Size Range. Unlike most competitors' kits, Norgen's Total RNA Purification Kit allows for the isolation of all sizes of RNA, from the very large RNA down to the microRNA without the use of phenol. Total RNA was isolated from 1 x 109 E. coli cells using Norgen’s Total RNA Purification Kit and a competitor’s kit. Five microliters and 1 µL of the 50 µL isolated RNA was analyzed on an agarose gel (Panel A) and the Agilent® 2100 BioAnalyzer RNA Nano 6000 chip (Panel B), respectively. Note the presence of small RNA species (red circle) in the samples isolated via Norgen's kit and the absence of these RNA species in the competitor RNA preparation.

Figure 2. Amplification of Both Large and Small RNAs in the Same Extraction. Norgen’s Total RNA Purification Kit isolates the complete size range of RNA, including small RNAs without the use of phenol. Total RNA was isolated from 0.75 million HeLa cells using Norgen’s Total RNA Purification Kit, various silica-based competitors and phenol-based TRI Reagent. RT-PCR was performed according to Shi and Chiang (2005). Fifteen microliters of the 50 µL isolated RNA were polyadenylated in a 50 µL Poly-(A)-Polymerase reaction. Four 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 qPCR reaction with primers against the human microRNAs (miR-19 and miR-21) and large mRNA (S15). Only the use of Norgen's Total RNA Purification Kit resulted in detection of both microRNAs and mRNA amplification products similar to TRI Reagent but without the use of phenol.

Figure 3. High Quality of RNA from a DiverseRangeof Inputs. Norgen’s Total RNA Purification Kit allows RNA isolation from a wide range of species or tissue types. Total RNA was isolated from 5 x 108 E. coli cells, 1 x 106 HeLa cells, 100 µL rat blood and 10 mg hamster kidney using Norgen’s Total RNA Purification Kit. One microliter of the 50 µL isolated RNA was analyzed on the Agilent® 2100 BioAnalyzer using an RNA Nano 6000 chip. Note the integrity of RNA from all inputs with the presence of small RNA species. Norgen’s Total RNA Purification Kit consistently isolates high quality RNA from various inputs that score a RIN value between 8 and 10.

Figure 4. Great Isolation Sensitivity. Norgen’s Total RNA Purification Kit allows sensitive RNA extraction from as little as a single cell. Total RNA was extracted from a decreasing number of 293 HEK cells from 1 million cells down to a single cell. Five microliters of the 50 µL isolated RNA was then subjected to a 20 µL reverse transcription using oligo dT primer. Three microliter of the reverse transcription was used in a 20 µL PCR reaction with primers to detect the human beta-actin transcripts. PCR products of beta-actin were detected from as little as a single cell. M is the marker lane.

Figure 5. Linear and Sensitive Isolation of Both Large and Small RNA. Norgen’s Total RNA Purification Kit allows consistent isolation of both large and small RNA from different input amounts. Total RNA was isolated from 10 to 100,000 HeLa cells using Norgen's Total RNA Purification Kit (blue), a competitor’s silica-based kit (green) and a phenol-based RNAextraction method (red). Relative expression of miR-21 (Panel A), and S15 (Panel B) was determined by RT-qPCR of total RNA samples. In brief, one microliter of the 50 µL isolated RNA was then subjected to a 20 µL reverse transcription using miR-21 stem-loop reverse primer or oligo dT primer. Two microliters of the reverse transcription was used in a 20 µL real-time PCR reaction with primers to detect the human miR-21 (Panel A) and the S15 transcripts (Panel B). The resulting threshold cycle (Ct) values were plotted against input cell number. RNA isolated using Norgen’s Total RNA Purification had the best linearity (higher R2) and sensitivity (lower Ct) for both large RNA (S15) and small RNA (miR-21).

Figure 6. Effective and Consistent Detection of miRNA from Plasma. Norgen's Total RNA Purification Kit can effectively isolate miRNA from plasma. Total RNA was isolated from 50, 100 or 200 µL of rat plasma in triplicates using Norgen's Total RNA Purification Kit (blue), a competitor’s silica-based kit (green) and a phenol-based RNAextraction method (red). Stem loop RT-qPCR using primers specific to miR-21 was performed. In brief, two microliters of the 50 µL isolated RNA was then subjected to a 20 µL reverse transcription using miR-21 stem-loop reverse primer or oligo dT primer. Three microliters of the reverse transcription was used in a 20 µL real-time PCR reaction with primers to detect the human miR-21. Norgen's Total RNA Purification Kit is the only product that showed (1) consistent detection of miR-21 transcripts across all input volumes, and(2) Ct values correlated to input volume (decrease Ct with increase input).

Figure 7. Recovering Diverse miRNA Species from Plasma with Better Consistency. Norgen’s Total RNA Purification Kit isolatesplasma RNAthat performs more consistently in downstream applications such as microarrays. Total RNA including miRNA was isolated from 100 µL of mouse plasma in duplicate using Norgen's Total RNA Purification Kit, Competitor A's leading miRNA Kit or a phenol-based RNAextraction method. One hundred nanograms of extracted RNAfrom each kit wasapplied to anIllumina microRNA expression profiling kit. Scatter plots display better consistency (better clustering) of replicate signals from Norgen's samples. Genes with Pval < 0.01 for both replicates were in blue. The associated table suggested that Norgen's protocol recovered the same diversity of miRNA with better consistency (higher r2value).

Figure 8: Better Diversity of miRNA Detected from Plasma. Norgen’s Total RNA Purification Kit isolates miRNA from plasma with better diversity than a leading competitor. Total RNA including miRNA was isolated from 100 µL of plasma using Norgen's Total RNA Purification Kit or 625 µL of plasma using Competitor A's leading miRNA Kit, and was applied to an NCode expression profiling kit. Microarray images suggested that Norgen’s Total RNA Purification Kit (left) isolates a better diversity of miRNA from smaller input amount of plasma thanthe competitor’s miRNA kit (right). Image courtesy of LCSciences, Houston. (www.lcsciences.com).

APPLICATIONS

Kit Specifications
Binding Capacity Per
Column
Up to 50 µg RNA
Maximum Loading Volume Per Spin Column
650 µL
Size of RNA Purified
All sizes, including < 200 nt
Time to Complete 10 Purifications
20 minutes
RNA Yield
Liver (10 mg)
30 µg
Kidney (10 mg)
10 µg
Brain (10 mg)
12 µg
Blood (hamster, 100 µL)
5 µg
HeLa (1 x 106)
15 µg
CHO (1 x 106)
11 µg
Yeast (1 x 108)
30 µg
E coli (1 x 109)
43 µg


Total RNA Purification Kit Contents

1. Lysis Solution
2. Wash Solution
3. Elution Buffer
4. Mini Spin Columns
5. Collection Tubes
6. Elution Tubes
7. Product Insert


Storage Conditions and Product Stability
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.