Systematic comparison of RNA extraction techniques from frozen and fresh lung tissues: checkpoint towards gene expression studies
- Equal contributors
1 Clinical Research Group "Chronic Airway Diseases", Faculty of Medicine, Philipps-University Marburg, Marburg, Germany
2 Dept. of Internal Medicine, Division of Nephrology, University Hospital Giessen & Marburg GmbH, Marburg, Germany
3 Dept. of Internal Medicine, Division of Respiratory Medicine, University Hospital Giessen & Marburg GmbH, Marburg Hessen, Germany
4 Section of Experimental Pneumology, Research Center Borstel, Leibniz Center for Medicine and Biosciences, Borstel, Germany
5 School of Biotechnology, Gautam Buddha University, Greater Noida – 201308, India
Diagnostic Pathology 2009, 4:9 doi:10.1186/1746-1596-4-9Published: 24 March 2009
The reliability of gene expression profiling-based technologies to detect transcriptional differences representative of the original samples is affected by the quality of the extracted RNA. It strictly depends upon the technique that has been employed. Hence, the present study aimed at systematically comparing silica-gel column (SGC) and guanidine isothiocyanate (GTC) techniques of RNA isolation to answer the question which technique is preferable when frozen, long-term stored or fresh lung tissues have to be evaluated for the downstream molecular analysis.
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.