1. Having Trouble Visualizing Your RNA Markers?

At Ambion, we believe the benefits of including Ethidium Bromide (EtBr) during Northern RNA analysis far outweigh any minor effects on mobility, transfer and hybridization. EtBr staining gives the scientist information about RNA sample integrity and facilitates the labeling of RNA marker and rRNA positions on the transfer membrane. Staining RNA with EtBr for visualization on formaldehyde- or glyoxal-agarose gels can be accomplished by one of three methods:

  1. Incorporating EtBr into the gel,
  2. Post-staining the gel with EtBr after electrophoresis or
  3. Adding EtBr to the gel loading buffer

The first two methods, although frequently used, are liberal in their use of EtBr, a powerful mutagen. Incorporating EtBr into the entire gel results in strong background fluorescence making it very difficult to discern RNA-specific staining. Post-staining is time consuming and may result in diffusion of the marker bands, so that sizing accuracy is lost. Adding EtBr to the gel loading dye prior to mixing with markers and samples is faster, uses less EtBr, eliminates background staining, and allows easy manipulation of the staining intensity. Ethidium bromide concentration and sample denaturation temperature are both positively correlated with the intensity of RNA fluorescence (see Figures 1 and 2). While high concentrations of EtBr may alter the rate of migration, decrease transfer efficiency and/or inhibit hybridization of RNA, here we show that EtBr concentrations up to 50 µg/ml do not significantly alter in mobility. Furthermore, transfer and hybridization efficiencies do not differ significantly from the post electrophoresis staining method (1). Based on these and other experiments done at Ambion, we recommend adding 30 µl/ml EtBr to Northern gel loading buffers prior to mixing with samples. The EtBr will give the researcher and opportunity to evaluate sample integrity and gel separation without compromising mobility, transfer and hybridization significantly. We also recommend heating samples to 80°C for 10 min. prior to loading for maximum staining intensity.

Figure 1. EtBr Concentration in Loading Buffer Affects Stain Intensity. Two µg of Ambion's RNA Millennium™ Markers were combined with three volumes of formaldehyde gel loading buffer to a final EtBr concentration of 10, 20, 30, 40 and 50 µg/ml for lanes 1, 2, 3, 4 and 5, respectively. Samples were heated at 70°C for 10 min. before loading on a 1% formaldehyde-agarose gel. The gel was run for 1.5 hr at 5 V/cm and photographed under UV light.


Figure 2. Denaturation Temperature Affects EtBr Stain Intensity.
Each lane was loaded with 2 ug of Ambion's RNA Millennium™ Markers in three volumes of formaldehyde gel loading buffer containing 30 µg/ml EtBr. Samples in lanes 1, 2, 3, 4, and 5 were preheated at 65, 70, 75, 80 and 85°C, respectively, before loading. Samples were run on a 1% formaldehyde-agarose gel at 5 V/cm for 1.5 hr and photographed under UV light..

Reference
Ogretmen, B., Ratajczak, H., Kats, A., and Stark, BC. (1993) Effects of staining of RNA with EtBr before electrophoresis on preformance of Northern blots. Biotechniques 14, 932-935.

2. Does Increasing Input Total RNA Affect Yield of RT-PCR Product?

Researchers performing reverse transcriptase-polymerase chain reaction (RT-PCR) to detect specific mRNA transcripts typically use 1 to 5 µg of total RNA in a 20 µl RT reaction (1). In the case of rare or less abundant mRNA messages, it may be advantageous to use greater RNA amounts in order to generate sufficient cDNA for PCR amplification. We investigated the effect of using increased amounts of total RNA from K562 (a human lymphocytic leukemic cell line) on the yield of Topoisomerase-II-ß (Top2ß) RT-PCR products. The human Top2ß coding region is approximately 5 kb in length (GenBank Accession #X68060).

Total RNA was isolated from K562 cells using Ambion's RNAqueous™ Kit, and DNase-treated with Ambion's DNA-free™ Reagents. The RNA was precipitated with ammonium acetate and ethanol, and resuspended in nuclease-free water to a concentration of 2 mg/ml. Increasing RNA amounts (1-20 µg) were then used in RT reactions using MMLV RNase H- Reverse Transcriptase. RT reactions were primed with either oligo dT or random decamers. Reactions were incubated for 1 hour at 42°C followed by heat inactivation for 10 minutes at 92°C. No template (-temp) and no Reverse Transcriptase (-RT) negative control reactions were included. Twenty µg of total RNA was used in the (-RT) control reactions.

Forward and Reverse primers specific to the 5'-end of the Top2ß mouse mRNA were chosen using primer design software. These primers contain several mismatches to the human Top2ß sequence, however there are no mismatches closer than four bases from the 3' ends. Interestingly, no difference in yield of amplification product was seen using the mouse Top2ß primers with mouse and human total RNA (data not shown). A constant amount (5 µl) from each 20 µl RT reaction was used in a 50 µl total volume PCR reaction performed with Ambion's SuperTaq™ thermostable polymerase. The expected size of the Top2ß PCR amplicon is 452 bp.

Figure 1 shows that for RT reactions primed with oligo dT, the PCR yields increased slightly initially, and then reached a plateau with additional amounts of RNA. No inhibition of RT-PCR was seen with increasing amounts of input RNA. Conversely, PCR yields slightly decreased at the higher RNA amounts for RT reactions primed with decamers. Overall, reactions primed with random decamers resulted in slightly greater PCR yields compared to RT reactions primed with oligo dT for this sequence. The total RNA prep did not contain any EDTA. (Note that the addition of large amounts of RNA containing EDTA may inhibit RT-PCR reactions due to chelation of magnesium ions.) This experiment suggests that while it is possible to increase input RNA levels without seriously inhibiting the RT-PCR reactions, little change in RT-PCR product is realized.


Figure 1. Effect of Increasing Amounts of Total RNA on RT-PCR Yields of Top2ß. RT reactions were done with either oligo dT or random decamers and 1 to 20 µg of K562 total RNA. Ten µl of each PCR reaction were run on a 2.5% native agarose gel in the presence of loading dye containing ethidium bromide. The gel was photographed using a digital image capturing system. The following profile was used in the PCR step: Heat 2 min., 94°C, Cycle 30X: 94°C, 20 sec.; 55°C, 20 sec.; 72°C, 30 sec.; Hold 7 min., 72°C.


References
  1. Powell, LM. 1990. RNA Processing: Apo-B. In PCR Protocols: A Guide to Methods and Applications. Chapter 29. (ed. Innis, M.A. et al.) Academic Press, San Diego, CA.
  2. Park, S. 1990. PCR in the Diagnosis of Retinoblastoma. In PCR Protocols: A Guide to Methods and Applications. Chapter 49. (ed. Innis, M.A. et al.) Academic Press, San Diego, CA.