Working with RNA in the laboratory demands precision and care. Whether you're new to the world of molecular biology or looking to refresh your knowledge, this quick guide will provide essential tips for RNA handling and precautions to ensure successful experiments. Let's dive in!
Mastering RNA Handling
Handling and Precautions for RNA
◼ Reagents used for RNA preparation and analysis should be kept separate from other reagents.
◼ During work, avoid unnecessary talking, wear a mask, and use clean disposable plastic gloves. Use dedicated RNA workstations and benches to minimize contamination.
◼ Prepare reagent solutions for RNA work with 0.1% DEPC-treated water and autoclave them before use. If autoclaving is not possible due to sensitive components in the reagents, prepare solutions using sterilized equipment and water, followed by filtration sterilization.
Method for preparing 0.1% DEPC-treated water:
Add Diethyl pyrocarbonate (DEPC) to distilled water at a concentration of 0.1% (v/v). Stir the solution at room temperature for one night (or 12 hours at 37°C). Then, autoclave the solution at 120°C for 30 minutes to remove DEPC completely.
Caution: DEPC is a chemical reagent used as an RNase inhibitor, but it is carcinogenic, so handle it with care.
◼ Most disposable sterile plasticware available in the market is RNase-free and can be used directly for experiments. However, autoclave standard microcentrifuge tubes and micro pipet tips before use. If using glassware or spatulas, autoclave them at 180°C for at least 1 hour. If autoclaving is not possible, soak them in 0.1% DEPC solution at room temperature for one night (or 12 hours at 37°C), then autoclave before use.
Preparation of RNA Samples
◼ In many experiments, high-purity RNA samples are required. Especially for DNA chip analysis, impurities such as carbohydrates or proteins can interfere with the reaction or lead to high background signals. Preventing contamination with genomic DNA is also crucial.
◼ For tissues and cells, RNA should be extracted immediately after sample collection. If immediate extraction is not possible, store samples at -80°C or in liquid nitrogen.
Preparation of Total RNA
Use methods such as cesium chloride density gradient centrifugation or Guanidine thiocynate phenol chloroform (AGPC) extraction, or commercially available RNA purification reagents.
Purification of Poly(A)+ RNA
Poly(A)+ RNA is commonly isolated from total RNA using methods involving Oligo(dT) Cellulose or similar techniques.
Purity and Concentration Assessment of RNA
◼ In most cases, the purity of RNA greatly affects experimental results. Therefore, it is essential to assess RNA purity before use.
◼ Purity Assessment of Total RNA by Gel Electrophoresis
Denature 1-2 µg of total RNA (65°C for 10 minutes) and electrophorese it on a 1% agarose gel (TBE buffer). Intact total RNA should show two distinct ribosomal RNA bands (28S and 18S in eukaryotes, 23S and 16S in prokaryotes) in a ratio of approximately 2:1. If ribosomal RNA bands are smeared, it may indicate RNA degradation due to RNase contamination. If there are larger bands than 28S (or 23S), it suggests the presence of genomic DNA contamination, and DNase I (RNase-free) treatment should be performed to remove genomic DNA.
◼ Purity and Quantification of Total RNA and Poly(A)+ RNA by UV Absorbance
Measure the absorbance at 260nm and 280nm to assess purity and concentration of total RNA (or Poly(A)+ RNA).
A260/A280 ratio:
A ratio of 1.8-2.1 indicates low protein contamination and high-purity RNA samples.
A ratio below 1.7 is not suitable for DNA chip experiments.
Calculate RNA concentration using A260=1, which corresponds to 40 µg/ml.
Example: If you have 100 µl of RNA sample, and the absorbance at A260 is 0.65, then
RNA concentration = 40 µg/ml x A260 x dilution factor
= 40 x 0.65 x 50
= 1300 µg/ml
Total RNA amount = concentration x sample volume (ml)
= 1300 x 0.1
= 130 µg
Precise analysis of RNA, including total RNA and mRNA, can be achieved through nucleic acid gel electrophoresis and automated detection devices for accurate results.
