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The success of many fundamental molecular biology experiments hinges on obtaining high quality, intact RNA
Different applications impose different requirements on RNA quality. RNA that will be used in RT-PCR should be completely free of genomic DNA contamination. The presence of even trace amounts of genomic DNA can generate false positive products amplified during the PCR. The most effective method of removing DNA from RNA preparations is to digest with DNase I certified free of contaminating RNase.
Ambion offers two RNA isolation kits specifically designed for RT-PCR applications — RNAqueous-4PCR and RNAqueous-96. Each kit employs Ambion's RNAqueous technology; a rapid, filter-based RNA isolation method that does not use phenol, chloroform or other toxic organic chemicals. The procedure consists of disrupting tissues or cells in a guanidinium-based lysis solution, followed by binding the RNA to a glass fiber filter, washing the filter to remove contaminants, and then eluting the RNA in a small volume (50 µl). Additionally, each kit includes a DNase treatment step using Ambion's DNase I that has been rigorously tested and is certified RNase-free. The resulting RNA is then ready for RT-PCR applications. The sections below describe each of these products.
The RNAqueous-4PCR Kit: DNA-free RNA Isolation For RT-PCR
The RNAqueous-4PCR Kit produces RNA that is completely free of genomic DNA contamination from samples as small as 1 mg or 100 cells. The kit is ideal for needle biopsies and small numbers of isolated cells. Three plastic pestles are included to aid in disruption of small amounts of tissue. The DNase treatment and removal reagents consist of RNase-free DNase I, 10X DNase I Buffer, and a novel reagent for rapid and safe removal of DNase I and divalent cations. The kit contains linear acrylamide, a coprecipitant used to concentrate the eluted RNA by alcohol precipitation. Each kit contains reagent for 30 RNA isolations. Total RNA isolated from 3 mg samples of various mouse tissues was used as template in RT-PCR (Figure 1).
Figure 1. Using Total RNA Isolated With RNAqueous-4PCR. 10% of several RNA preparations were used for reverse transcription. 10% of the resulting cDNA was amplified by PCR using S15 primers. The lanes to the left of the markers are minus-RT control PCRs, demonstrating the lack of genomic DNA contamination in these RNA samples. The lanes to the right of the markers show the S15 product from the indicated samples.
Figure 1. Using Total RNA Isolated With RNAqueous-4PCR. 10% of several RNA preparations were used for reverse transcription. 10% of the resulting cDNA was amplified by PCR using S15 primers. The lanes to the left of the markers are minus-RT control PCRs, demonstrating the lack of genomic DNA contamination in these RNA samples. The lanes to the right of the markers show the S15 product from the indicated samples.
The RNAqueous-96 Kit: High-throughput, 96-well RNA Isolation for RT-PCR
The RNAqueous-96 Kit is ideal for isolation of total RNA from multiple, small samples. The procedure is performed in 96-well filter plates included in the kit and can be used with either vacuum filtration or centrifugation methods (the 96-well plates are compatible with most common 96-well plate vacuum manifolds and 96-well plate centrifuge rotors). Both methods provide high yields of intact RNA. DNase treatment is done on the filter, after the RNA is bound. Following the DNase digestion, DNase I is washed away and the RNA is eluted.
As demonstrated by the experiment described in Table 1, the RNAqueous-96 Kit provides consistent yields of RNA from various amounts of starting material. The high quality of RNA isolated with the RNAqueous-96 Kit is illustrated in Figure 2. Figure 3 shows TaqMan (ABI PRISM, 7700) quantitative RT-PCR data for ß-actin with RNA from the same samples that are shown in Figure 2. The RNAqueous-96 Kit includes reagents and supplies for 192 RNA isolations.
Table 1. RNA Yields from Varying Numbers of Cells. The RNAqueous-96 Kit was used to isolate total RNA from varying numbers of cultured HeLa cells as indicated in the above chart. Samples were filtered by centrifugation using the Beckman Spinchron DLX centrifuge with the PTS 2000 rotor and appropriate microplate carriers. Yield and purity of the recovered RNA was measured using a Beckman DU 660 spectrophotometer. Each value in the chart above represents the average of 48 replicates. (Data courtesy of Dr. Jim Collins, Beckman Instruments)
Figure 2. RNA Isolated from K562 Leukemic Cells Using the RNAqueous-96 Kit. Cultured K562 leukemic cells were lysed to a concentration of 3.3 x 106 cells/ml and aliquots of the indicated numbers of cells were processed in triplicate with the RNAqueous-96 Kit. 1/10th of each preparation was analyzed on an EtBr-stained denaturing agarose gel. Table 1. RNA Yields From The RNAqueous-96 Kit. Cultured HeLa cells were subjected to the RNAqueous-96 Kit procedure. Yield and purity of the recovered RNA was measured using a Beckman DU 660 spectrophotometer. Each value in the chart above represents the average of 48 replicates. (Data courtesy of Dr. Jim Collins, Beckman Instruments)
Figure 3. Real-time RT-PCR Using Total RNA Isolated Using the RNAqueous-96 Kit. 1/10th of the K562 leukemic cell RNAs isolated as described in Figure 2 were used as templates in 20 µl reverse transcription reactions. 5 µl of each RT reaction were then used as template in 50 µl real-time PCR experiments using an ABI PRISM(r) 7700 instrument with primers and a FAM-labeled probe for ß-actin (PE Applied Biosystems). The sample represented on the far right is the -RT control. ABI PRISM(r) 7700 is a registered trademark of Perkin Elmer.
As demonstrated by the experiment described in Table 1, the RNAqueous-96 Kit provides consistent yields of RNA from various amounts of starting material. The high quality of RNA isolated with the RNAqueous-96 Kit is illustrated in Figure 2. Figure 3 shows TaqMan (ABI PRISM, 7700) quantitative RT-PCR data for ß-actin with RNA from the same samples that are shown in Figure 2. The RNAqueous-96 Kit includes reagents and supplies for 192 RNA isolations.
| # of Cells | A260/A2800 | RNA Yield (µg) |
|---|---|---|
| 1 x 105 | 2.09 +/- 0.02 | 1.49 +/- 0.06 |
| 5 x 105 | 2.03 +/- 0.03 | 6.88 +/- 0.47 |
| 1 x 106 | 2.02 +/- 0.01 | 14.87+/- 0.71 |
| 2 x 106 | 1.90 +/- 0.01 | 24.32 +/- 1.67 |
Table 1. RNA Yields from Varying Numbers of Cells. The RNAqueous-96 Kit was used to isolate total RNA from varying numbers of cultured HeLa cells as indicated in the above chart. Samples were filtered by centrifugation using the Beckman Spinchron DLX centrifuge with the PTS 2000 rotor and appropriate microplate carriers. Yield and purity of the recovered RNA was measured using a Beckman DU 660 spectrophotometer. Each value in the chart above represents the average of 48 replicates. (Data courtesy of Dr. Jim Collins, Beckman Instruments)
Figure 2. RNA Isolated from K562 Leukemic Cells Using the RNAqueous-96 Kit. Cultured K562 leukemic cells were lysed to a concentration of 3.3 x 106 cells/ml and aliquots of the indicated numbers of cells were processed in triplicate with the RNAqueous-96 Kit. 1/10th of each preparation was analyzed on an EtBr-stained denaturing agarose gel. Table 1. RNA Yields From The RNAqueous-96 Kit. Cultured HeLa cells were subjected to the RNAqueous-96 Kit procedure. Yield and purity of the recovered RNA was measured using a Beckman DU 660 spectrophotometer. Each value in the chart above represents the average of 48 replicates. (Data courtesy of Dr. Jim Collins, Beckman Instruments)
Figure 3. Real-time RT-PCR Using Total RNA Isolated Using the RNAqueous-96 Kit. 1/10th of the K562 leukemic cell RNAs isolated as described in Figure 2 were used as templates in 20 µl reverse transcription reactions. 5 µl of each RT reaction were then used as template in 50 µl real-time PCR experiments using an ABI PRISM(r) 7700 instrument with primers and a FAM-labeled probe for ß-actin (PE Applied Biosystems). The sample represented on the far right is the -RT control. ABI PRISM(r) 7700 is a registered trademark of Perkin Elmer.