For sequential isolation of total RNA, genomic DNA and total proteins from the same sample
Norgen's RNA/DNA/Protein Purification Plus Micro Kit provides a rapid method for the isolation and purification of total RNA, genomic DNA and proteins sequentially from a single sample of cultured animal cells, small tissue samples, microdissected samples including laser-capture microdissection (LCM), blood, fungi or plants. The total RNA, genomic DNA and proteins are all column purified in less than 30 minutes. The RNA and DNA can be eluted in as little as 20 µL while the protein can be eluted in as little as 50 µL. This kit is ideal for researchers who are interested in studying the genome, proteome and transcriptome of a single sample, such as for studies of microRNA profiling, gene expression including gene silencing experiments or mRNA knockdowns, studies involving biomarker discovery, and for characterization of cultured cell lines. Norgen's RNA/DNA/Protein Purification Plus Micro Kit is especially useful for researchers who are isolating macromolecules from precious, difficult to obtain or small samples such as biopsy materials, LCM or single foci from cell cultures, as it eliminates the need to fractionate the sample. Furthermore, analysis will be more reliable since the RNA, DNA and proteins are derived from the same sample, thereby eliminating inconsistent results.
Panel A is a 1X MOPS 1% agarose gel showing the RNA that was isolated from 2 different samples of ~500,000 HeLa cells using either Norgen's RNA/DNA/Protein Purification Plus Micro Kit or a competitor's multiple-analyte purification kit. Both kits isolated large RNA (represented by 28S and 18S rRNA) with high integrity but Norgen's RNA/DNA/Protein Purification Plus Micro Kit provided the added benefit of recovering small RNA without any additional protocols. Panel B is a result from a bioanalyzer resolution of the eluted RNA. Similar to the agarose gel, Norgen's RNA/DNA/Protein Purification Plus Micro Kit showed the added benefit of recovering small RNA as well as a much higher concentration of RNA (Panel C). The difference in small RNA recovery was also demonstrated by gene-specific RT-qPCR. Two microliters of RNA isolated from both 500,000 and 3,000 HeLa cells were used in RT-qPCR reactions for human S15 (for Large RNA) and miR-21 (for microRNA) genes. The RNA isolated by Norgen's RNA/DNA/Protein Purification Plus Micro Kit showed similar or better (lower) Ct value than RNA isolated by the competitor's kit for the large RNA. More importantly, Norgen's RNA/DNA/Protein Purification Plus Micro Kit showed superior recovery of small RNA including microRNAs as depicted by the miR-21 RT-qPCR (Panel D).
Panel A is a 1% agarose gel showing equal amounts of gDNA isolated from the same HeLa cells using Norgen's RNA/DNA/Protein Purification Plus Micro Kit or competitor's multiple-analyte purification kit. Lane M is Norgen's HighRanger 1 kb DNA Ladder. The gel shows high quality, intact genomic DNA with a better DNA concentration using Norgen's RNA/DNA/Protein Purification Plus Micro Kit (Panel B). Panel C is the result of qPCR amplification of 2 µL of eluted genomic DNA using 5S rRNA-specific primers. Genomic DNA isolated using Norgen's RNA/DNA/Protein Purification Plus Micro Kit is of high quality, and performed better than DNA purified using a competitor's product.
Norgen's RNA/DNA/Protein Purification Plus Micro Kit provides an additional column purification step for effective concentration and clean-up of the isolated proteins. In contrast, most competing multiple analyte isolation products require protein precipitation and the precipitated proteins need to be resuspended in a buffer with high-detergent content (such as SDS-PAGE loading dye) for full recovery. Protein fractions (from hamster liver) isolated by Norgen's RNA/DNA/Protein Purification Plus Micro Kit and a competitor's kit were resolved on a 12% SDS-PAGE protein gel. Panel A showed that when the competitor's precipitated protein fraction was resuspended in a provided SDS-PAGE loading buffer, the protein recovery was similar among the two kits. Panel B showed that when the same precipitated protein fraction from the competitor's kit was resuspended in a Tris-based buffer containing no detergent or denaturant, the protein recovery became drastically reduced. In contrast, Norgen's RNA/DNA/Protein Purification Plus Micro Kit purified proteins by column and the eluted proteins are already in a buffer compatible with most downstream applications including mass spectrophotometry as well as standard protein quantification methods (including Bradford assays).
|Maximum Column Binding Capacity||35 µg for RNA, 10 µg for DNA, 100 µg for protein|
|Maximum Column Loading Volume||650 µL|
|Size of RNA Purified||All sizes, including small RNA (<200 nt)|
|Size of DNA Purified||≥ 30 kb|
|Maximum Amount of Starting Material||Animal Cells: 5 x 105 cells
Animal Tissues: 5 mg (for selected tissues)
Blood: 50 µL
Plant, Fungi Tissues: 25 mg
|Time to Complete 10 Purifications||30 minutes|
|Average Yields||HeLa Cells (5 x 105 cells):
7.5 µg RNA
1-2 µg DNA
70 µg protein
|RNA/DNA/Protein Purification Plus Micro Kit||51600||Room Temp.||50 preps||595.00||Buy|