Systematic comparison of RNA extraction techniques from frozen and fresh lung tissues: checkpoint towards gene expression studies

Frozen lungs (n = 3) were prepared by long-term storage (2.5 yrs) in -80°C while fresh lungs (n = 3) were harvested and processed immediately. The purity and quantification of RNA was determined with a spectrophotometer whereas the total amounted copy numbers of target sequences were determined with iCycler detection system for assessment of RNA intactness (28S and 18S) and fragment sizes, i.e. short (GAPDH-3' UTR), medium (GAPDH), and long (PBGD) with 200 bp, 700 bp, and 1400 bp distance to the 3'ends of mRNA motif, respectively.Total yield of RNA was higher with GTC than SGC technique in frozen as well as fresh tissues while the purity of RNA remained comparable. The quantitative reverse transcriptase-polymerase chain reaction data revealed that higher mean copy numbers of 28S and a longer fragment (1400 bp) were obtained from RNA isolated with SGC than GTC technique using fresh as well as frozen tissues. Additionally, a high mean copy number of 18S and medium fragment (700 bp) were obtained in RNA isolated with SGC technique from fresh tissues, only. For the shorter fragment, no significant differences between both techniques were noticed.Our data demonstrated that although the GTC technique has yielded a higher amount of RNA, the SGC technique was much more superior with respect to the reliable generation of an intact RNA and effectively amplified longer products in fresh as well as in frozen tissues.DNA-microarray and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) are two powerful techniques widely used in functional genomics for the analysis of gene expression profiles. These techniques are highly dependent on the quality of total RNA [1-3] obtained from either fresh or frozen biological samples.RNA is a particularly labile bio-molecule and is much more susceptible to degradation by endogenous- and exogenous-nucleases and to non-specific degradation by divalent cations, heat, elevations in pH, and storage of tissue or cells over extende