Catalog No. : MN-740955.250
NucleoSpin RNA (250)
|NucleoSpin RNA (250)||
NucleoSpin RNA® Columns with Collection Tubes, Collection Tubes (2 mL), Collection Tubes (1.5 mL), NucleoSpin® Filters, buffers, RNase-free rDNase
NucleoSpin® RNA is recommended for isolation of total RNA from cultured cells, tissue, bacteria, yeast, cell-free biological fluids, and reaction mixtures. This kit allows purification of up to 70 μg of highly pure, DNA-free total RNA. As reference RNA from 106 cultured HeLa cells is prepared following the standard protocol resulting in 15 to 20 μg, but at least 10 μg of total RNA. It is possible to detect transcripts from very low amounts of cells.
Total RNA prepared with NucleoSpin® RNA is suitable for applications like reverse transcriptase-PCR (qRT-PCR), Northern blotting, primer extension, array technology or RNase protection assays. For isolation of high-quality RNA it is important to prevent degradation of the RNA and to eliminate genomic DNA. With the NucleoSpin® RNA method, cells are lysed by incubation in a solution containing large amounts of chaotropic ions (see procedure). This lysis buffer immediately inactivates RNases – which are present in virtually all biological materials – and creates appropriate binding conditions which favour adsorption of RNA to the silica membrane. After lysis, homogenization and reduction of viscosity are achieved by filtration with NucleoSpin® Filter units provided with the kit.
NucleoSpin® RNA procedure
Contaminating DNA bound to the silica membrane is removed by a rDNase solution which is directly applied onto the silica membrane during the preparation. Optimal conditions for the rDNase are achieved by washing the silica membrane with a specific desalting buffer before treatment (see Application data). Salts, metabolites and macromolecular cellular components are removed by simple washing steps with two different buffers. Total RNA is finally eluted with RNase-free water supplied with the kit. A260/A280 is between 1.9 and 2.1. High structural integrity is shown by sharp rRNA bands (see Application data). Treatment of RNA eluates with RNase A shows efficient removal of genomic DNA in NucleoSpin® RNA preparations, whereas a clear distinct DNA band is visible in RNA samples prepared with the kit of competitor Q (see Application data). Truly quantitative removal of genomic DNA is shown by PCR control experiments using different sensitive gene-specific primers.
Hands-on time for total RNA preparation from cultured cells or tissue with NucleoSpin® RNA is less than 30 minutes.
High recoveries permit detection of a transcript from as few as 10 HeLa cells by RT-PCR
RT-PCR of total RNA isolated with the NucleoSpin® RNA standard protocol from 105, 104, 103, 330, 100, 33, 10, 3 and 0 HeLa cells (1–9). The RT-PCR was done with 3 μL of total RNA each (elution volume: 40 μL) and ß-actin primers. The specific RT-PCR product is 626 bp of size.
Optimized rDNase digest by use of MDB (membrane desalting buffer)
Total RNA was purified using NucleoSpin® RNA with (+ MDB) or without (– MDB) use of membrane desalting buffer prior to rDNase digest. DNA contamination of purified RNA samples was determined by LightCycler™ analysis (A).
Corresponding PCR products were separated by agarose gel electrophoresis (B).
No genomic DNA is detected if MDB is applied.
DNA removal you can see
A: Total RNA was purified from 106 HeLa cells using the NucleoSpin® RNA kit in comparison to competitor Q. 10 μL of each eluate (elution volume 50 μL) were analyzed on a 1.2 % formaldehyde gel. The A260/A280 was on average 2.1.
Due to lower detection sensitivity of genomic DNA in a denaturing formaldehyde gel contaminating genomic DNA is often not visible.
B: For visualization of residual genomic DNA 10 μL of each eluate were treated with RNase A and subsequently loaded onto a 1 % TAE agarose gel. The NucleoSpin® RNA kit contains rDNase for on-column DNA digestion.
M: λHindIII, MBI-Fermentas
Optimized rDNase treatment eliminates genomic DNA in NucleoSpin® RNA preparations, whereas a clear distinct band of contaminating genomic DNA is visible in RNA samples prepared with a kit of competitor Q.
High-quality total RNA from hard-to-lyse bacterial strains
A: Formaldehyde agarose gel of total RNA purified with the NucleoSpin® RNA kit. Total RNA was isolated from Pseudomonas putida (1, 2) and Paenibacillus polymyxa (4, 5), (3, 6: RNA ladder as marker).
Data kindly provided by S. Meier-Bethke, Federal Biological Research Center for Agriculture and Forestry, Institute for Plant Virology, Microbiology and Biosafety, Braunschweig, Germany
High-quality total RNA from hard-to-lyse bacterial strains
B: 1.2 mL from a LB culture of Pseudomonas putida* (OD600 0.9) has been pelleted and resuspended in 100 μl TE-Buffer containing 0.05, 0.2, 0.8 mg/mL lysozyme, respectively. Incubation was performed at 37 °C for 10 min. The further isolation procedure was carried out according to the standard protocol of NucleoSpin® RNA. 6 μL of the eluate (elution volume 60 μL) was analyzed on a 1.2 % formaldehyde gel. The A260/A280 was on average 2.1.
*Due to a complex outer membrane Pseudomonas putida is known as a hard-to-lyse strain.
Even from hard-to-lyse bacterial strains high-quality total RNA can be isolated.
Quantitative DNA removal
PCR control experiment using purified total RNA (see Fig. 3.3 A above) as template. PCR reactions were performed for 30 cycles using gene-specific primers of different sensitivity regarding detection of genomic DNA. Amount of used template was 2 μL of the total RNA eluate. 30 μL of each PCR reaction were electrophoresed on a 1 % TAE agarose gel and stained with EtBr.
For best results with the BD Atlas™ Array BD/Clontech recommends the NucleoSpin® RNA kit.
Using the NucleoSpin® RNA kit nearly no DNA is detected even if primers (Gamma Actin) specific for a small and multi copy target are used. In comparison, with all primer systems DNA could be detected with the kit of competitor Q.
Purification of total RNA isolated with TRIzol® using the NucleoSpin® RNA kit
Total RNA from crushed dog heart tissue was isolated using TRIzol® with guanidinium thiocyanate and ß-mercaptoethanol. To every sample of dog heart 1.25 mL TRIzol® solution were added. The entire content of the tube was homogenized using an Omnihomogenizer for 30 to 45 s. An aliquot of 1.25 mL was transferred into each of twelve 2-ml tubes. 6 out of 12 samples were centrifuged for 5 min at 3.500 x g (prespin), the supernatants were transferred to new tubes. After adding chloroform to all 12 samples and phase separation each solution was purified using the NucleoSpin® RNA kit.
Yield, concentration, and purity were determined for all 12 samples. NucleoSpin® RNA preparations show a consistently high yield, concentration, and purity A260/A280 (with prespin: 2.05±0.02; without prespin: 2.07±0.03).
Total RNA isolated with TRIzol® can be purified with the NucleoSpin® RNA kit resulting in consistently high yield, concentration, and purity. A prespin of samples does not increase yield, concentration or purity.