|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
A column-based rapid method for the simultaneous isolation of DNA, RNA, miRNA and proteins
Sandeep K. Rajput*, Vivek P. Dave*†, Ankita Rajput‡, Hausila P. Pandey*, Tirtha K. Datta‡ and Rakesh K. Singh*1
*Department of Biochemistry, Banaras Hindu University, Varanasi, India, †Amity University, Noida, Uttar Pradesh, India, and ‡Animal Genomics Lab, ABTC, NDRI, Karnal 132001, India
In the 21st century, systems biology is a holistic approach to understand life by the cross-talk study between the genome, Rnome and proteome of a cell. We describe a column-based rapid method for the simultaneous extraction of DNA, RNA, miRNA (microRNA) and proteins from the same experimental sample without prior fractionation, which allows a direct correlation between genomic, epigenomic, transcriptomic and proteomic data. This method provides a simple and effective way to analyse each of these biomolecules without affecting yield and quality. We also show that isolated biomolecules are of the highest purity and compatible for all the respective downstream applications, such as PCR amplification, RT–PCR (reverse transcription–PCR), real-time PCR, reverse Northern blotting, SDS/PAGE and Western blot analysis. The buffers and reagents used in this method are optimized extensively to achieve the cost effective and reliable procedure to separate the functional biomolecules of the cell.
Key words: guanidine thiocynate, miRNA, nucleic acid isolation, protein, real-time PCR, reverse Northern blotting
Abbreviations: DIG, digoxigenin, GAPDH, glyceraldehyde-3-phosphate dehydrogenase, GuSCN, guanidine thiocyanate, miRNA, microRNA, RCF, relative centrifugal force, RT–PCR, reverse transcriptase–PCR, qRT–PCR, quantitative RT–PCR, RWB, RNA wash buffer, snRNA, small nuclear RNA
1To whom correspondence should be addressed (email firstname.lastname@example.org).
Received 11 June 2011/16 March 2012; accepted 3 May 2012
Published as Cell Biology International Immediate Publication 3 May 2012, doi:10.1042/CBI20110342
© The Author(s) Journal compilation © 2012 International Federation for Cell Biology