EFFECT OF HOMEMADE RNALATER ON THE QUALITY OF RNA
Extracting intact, high-quality RNA is the first and most critical step in performing molecular biology experiments. High-quality RNA is required for performing the quantitative and qualitative analysis of mRNA expression by RT-PCR, Northern blot hybridization and several nuclease protection assays. RNA isolation from tissue and cell is more challenging. Because these samples rich in endogenous ribonuclease (RNase). It is necessary to minimize the RNase activity to obtain high-quality RNA. So those tissues must be quickly homogenized in a strong protein denaturant usually guanidinium isothiocyanate, or it should be rapidly frozen in liquid nitrogen. Quick freezing and homogenization are even less convenient in the field. So there is a need for the reagent or method that allow preserving the high-quality, intact RNA from tissue sample at ambient temperature. Now a days RNAlater is commercially manufactured by many companies. RNAlater permeates tissues and protects the cellular RNA insitu for 1day at 37°C, one week at 25°C and one month at 4°C and these tissues can also be stored at -20°C for long term usage. However, the cost of commercial RNAlater (C. RNAlater) is higher. So, the protocol for preparing Homemade RNAlater (H. RNAlater) is evolved. The present study was conducted to test the effect of H. RNAlater and C. RNAlater on the quality of the RNA. Moreover, we have also tested the efficiency of Homemade Trizol (H. Trizol) and Commercial Trizol (C.Trizol) on the integrity of RNA.
RNAlaterTM RNA Preservation Medium (H. RNAlater) was prepared by following the method of Lader (2001). 200 mg of brain sample from Pangasius hypophthalmus was collected, of which 100 mg was immersed in 1 ml of H. RNAlater reagent and another 100 mg was immersed in 1 ml of C. RNAlater solution and preserved at 4°C. The next day total RNA was isolated from the preserved tissue samples using TRIzol® reagent (Invitrogen, USA) and also by H. Trizol following Sambrook et al. (2001) with slight modifications.
The results indicated that the RNA isolated by using TRIzol® reagent (Invitrogen, USA) from the tissue which preserved in H. RNAlater and C. RNAlater (Invitrogen, USA) gave high quality, intact RNA. But, the integrity of RNA isolated by using H. Trizol was lost. Hence, the result concluded that H. RNAlater is the best alternative to the C. RNAlater.
