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!
Handling and Precautions for RNA
- Isolate Reagents: Reagents used for RNA preparation and analysis should be kept separate from other reagents to prevent RNase contamination.
- Hygiene: During work, avoid unnecessary talking, wear a mask, and use clean disposable plastic gloves. Use dedicated RNA workstations and benches.
- Water Preparation: Prepare reagent solutions with 0.1% DEPC-treated water and autoclave them. If autoclaving is impossible, use sterilized equipment and perform 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 overnight (or 12 hours at 37°C).
- Autoclave the solution at 120°C for 30 minutes to remove residual DEPC completely.
- Plasticware & Glassware: Most disposable sterile plasticware is RNase-free. However, standard microcentrifuge tubes and pipette tips should be autoclaved. Glassware or spatulas should be baked at 180°C for at least 1 hour or soaked in 0.1% DEPC solution overnight before autoclaving.
Preparation of RNA Samples
High-purity RNA is crucial, especially for DNA chip analysis where impurities (carbohydrates, proteins) can cause high background signals. Preventing genomic DNA contamination is also vital.
- Immediate Extraction: Extract RNA immediately after sample collection from tissues or cells.
- Storage: If immediate extraction isn't possible, store samples at -80°C or in liquid nitrogen.
Preparation Methods
- Total RNA: Use Cesium chloride density gradient centrifugation, AGPC (Guanidine thiocyanate-phenol-chloroform) extraction, or commercial RNA purification kits.
- Poly(A)+ RNA: Commonly isolated from total RNA using Oligo(dT) Cellulose methods.
Purity and Concentration Assessment of RNA
Assessing RNA purity before experiments is essential as it directly impacts results.
1. Purity Assessment by Gel Electrophoresis
Denature 1-2 µg of total RNA (65°C, 10 min) and run on a 1% agarose gel.
- Intact RNA: Shows two distinct bands (28S and 18S) in a ~2:1 ratio.
- Degraded RNA: Ribosomal bands appear smeared.
- gDNA Contamination: Bands larger than 28S suggest genomic DNA presence (treat with RNase-free DNase I).
2. Purity and Quantification by UV Absorbance
Measure absorbance at 260nm (A260) and 280nm (A280).
A260/A280 Ratio Interpretation:
- 1.8 ~ 2.1: High purity (suitable for experiments).
- Below 1.7: Not suitable for sensitive experiments like DNA chips (protein contamination).
Calculation Example
Formula: Concentration = 40 µg/ml × A260 × Dilution Factor
(Assumption: A260=1 corresponds to 40 µg/ml RNA)
Scenario: 100 µl sample, A260 = 0.65, Dilution = 50x
1. Concentration = 40 × 0.65 × 50 = 1,300 µg/ml
2. Total Amount = 1,300 µg/ml × 0.1 ml = 130 µg
For the most accurate results, consider using automated detection devices (e.g., Bioanalyzer) alongside nucleic acid gel electrophoresis.
