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Total RNA Purification Kit for cultured animal cells, tissue samples, blood, plasma, serum, saliva, bacteria, yeast, fungi, plants and viruses Add to Cart

Cat#:	17200-NB
Quantity:	50 preps
Price:	313 € Special 281.70 € - valid until 30 September 2019
Supplier:	Norgen
Shipping:	RT

User Manual

Please note: This kit can be used instead of discontinued #53000-NB

• Isolate total RNA, from large rRNA down to microRNA (miRNA)
• Isolate high quality total RNA with complete size range from a variety of sources, even from plasma and serum
• RNA can be isolated and detected from as little as a single animal cell (see application note - link below)
• No phenol or chloroform extractions
• Rapid spin column format, protocol completed in <20min
• Kits for large and small input amounts and 96-well kit for high throughput applications also available

For large serum and plasma input amounts (1 ml - 5ml) please use the Plasma/Serum Circulating RNA Purification Kit (see link below).

Norgen´s Total RNA Purification Kit provides a rapid method for the isolation and purification of total RNA from cultured animal cells, small tissue samples, blood, serum/plasma, bacteria, yeast and fungi. 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 cellular components such as proteins, 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 real time PCR, reverse transcription PCR, Northern blotting, RNase protection and primer extension, and expression array assays.

Purification Technology
Purification is based on spin column chromatography using Norgen´s silicon carbide (SiC) resin as the separation matrix.
The process involves first lysing the cells or tissue of interest with the provided Lysis Solution. Binding solution and ethanol are then added to the lysate, and the solution 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 and genomic DNA will be removed in the flowthrough or retained on the top of the resin. The bound RNA is then washed twice with the provided wash buffer in order to remove any remaining impurities, and the purified total RNA is eluted with the elution buffer. The purified RNA is of the highest integrity, and the complete profile of RNA species is represented.

Silicon carbide-based RNA purification isolates a complete profile of RNA species enabling microRNA detection by qRT-PCR at low Ct values

Total RNA was isolated from HeLa cells using RNA isolation kits with spin column formats based on silicon carbide (SiC; Norgen), silica fiber (SiF) or silica matrix (SiM). Guanidinium thiocyanate/ phenol-based TRI Reagent isolation was used as positive control for isolation of the complete size range of RNA.
(A) Equal portions of the isolated RNA were resolved on an 8% Urea-PAGE gel.
(B) The relative expression of miR-21, miR-19, and S15 rRNA was determined by qRT-PCR, the resulting threshold cycle (Ct) values are shown.

High Quality of Isolated RNA with Complete Size Range

Unlike most competitor´s kits (lanes C and D), Norgen´s Total RNA Purification Kit (lane A and B) allows for the isolation of all sizes of RNA, from the very large RNA down to the microRNA. Lane M is Norgen´s RNA Ladder. Total RNA, large and small RNA species, can be seen in lanes A and B (Norgen).

Total E. coli RNA analyzed on the Agilent® 2100 BioAnalyzer. Note the presence of small RNA species (red circle) in the samples isolated via Norgen´s kit and the absence of these RNA sp. in the comptitor RNA preparation.

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 micrRNAs and mRNA amplification products similar to TRI Reagent but without the use of phenol.

High Quality of RNA from a Diverse Range of Inputs, even from Plasma

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.

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 RNA extraction 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).

Recovering Diverse miRNA Species from Plasma with Better Consistency. Norgen’s Total RNA Purification Kit isolates plasma RNA that 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 RNA extraction method. One hundred nanograms of extracted RNA from each kit was applied to an Illumina 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 r2 value).

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 than the competitor’s miRNA kit (right). Image courtesy of LC Sciences, Houston.

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 uL isolated RNA was then subjected to a 20 uL reverse transcription using oligo dT primer. Three microliter of the reverse transcription was used in a 20 uL 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, while the numbers correspond to the number of 293 HEK cells that were used for the RNA isolation procedure. Please view Application Note (see link below).

Typical RNA Yields

Liver (10 mg) 30 ug
Kidney (10 mg) 10 ug
Brain (10 mg) 12 ug
Blood (Hamster, 100 uL) 5 ug
HeLa (1 x 10e6) 15 ug
CHO (1 x 10e6) 11 ug
Yeast (1 x 10e8) 30 ug
E coli (1 x 10e9) 43 ug

Applications

• 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
• miRNA, also from plasma and serum, for discovery
• microRNA cloning and amplification
• PCR-based virus detection
• PCR-based viable bacteria detection
• Northern Blotting
• RNase Protection
• Primer Extension