Full Spectrum RNA Amplification.
How Does It Work?
The Full Spectrum Complete Transcriptome RNA Amplification Kit makes use of a specially developed Universal Primer Mixture that uniformly amplifies gene transcripts using low-cycle PCR. This approach maintains the relative levels of each transcript in the starting mRNA samples;even when using starting amounts of RNA as low as 5 ng. The Full Spectrum approach is also faster, requires fewer steps, and is more convenient than other techniques, including T7-based in vitro transcription, SMART cDNA amplification, or Ribo-SPIA amplification.
The Full Spectrum protocol entails just two steps:
- Universal Primer Binding and Synthesis of first-strand cDNA. The Universal Primer preferentially binds to all regions of messenger RNA and then initiates synthesis of the complementary DNA strand. This step takes approximately 1 hour. Remaining Universal Primer arbitrarily binds to the first-strand cDNA to prime second-strand synthesis in the following step.
- Second-strand cDNA synthesis and amplification. After an initial 5 min incubation to synthesize the second-strand, cycling commences to amplify the cDNA template. After 1 hour amplification, you have amplified template—ready for gene-specific PCR.
The amplified product can be used directly for quantitative PCR using primers specific for any gene sequence. No purification is required.
Amplify transcript 5’ ends from Degraded RNA: Full Spectrum vs. T7 IVT
To demonstrate the power of the Full Spectrum amplification method on degraded RNA, a partially degraded sample of human universal RNA and a control intact RNA sample were subjected to amplification with both the Full Spectrum method and a competitor’s T7 IVT kit. As expected for both amplification methods, the size distribution of the amplified RNA/cDNA was smaller for the partially degraded RNA (Figure 1A).
The amplified cRNA/cDNA was then interrogated by RT-PCR. For the T7 IVT method, the DNA template was generated by reverse transcription with random 9-mer primers. The amplified cDNA obtained by the SBI method was directly taken for PCR analysis. Sets of PCR primers were designed to amplify 3 regions of the human transferrin receptor (5.0 kb) and 2 regions of the human phospholipase A1 mRNA (1.75 kb). A map indicating the locations of the PCR primers is shown in Figure 1B. As a positive control, a primer pair was designed toward the 3’-end of the human β-actin mRNA (1.2 kb).
Figure 1A. Gel electrophoresis of intact and partially degraded RNA. The positions of the 18S and 28S ribosomal RNA bands are indicated.
Amplified cDNA obtained using the Full Spectrum method with intact RNA generated PCR products representing all 3 regions of the transferrin receptor, both regions of the phospholipase A1 and the β-actin control mRNAs. Identical results were obtained with partially degraded RNA, demonstrating the amplification of transcript 5’ ends by the Full Spectrum method (Figure 1B).
The results obtained with the T7 IVT method were quite different. In this case, strong bands were produced only with the control mRNA and the 3’-most regions of the test mRNAs with intact RNA. This also held true for partially degraded RNA (Figure 1C). It is clear from these experiments that the Full Spectrum method maintains mRNA sequence information all along the human transferrin receptor and phospholipase A1 mRNAs. The results illustrate the extreme 3’-end bias of the T7 IVT method, a flaw eliminated by the unique priming method used in SBI’s Full Spectrum Complete Transcriptome RNA Amplification Kit.
Make Quantitative RT-PCR templates with Full Spectrum Amplification Technology
Measuring the gene expression levels from small RNA samples is a very important technique in answering today’s functional genomics questions. Unfortunately, small sample quantities can limit the number of genes one can analyze by quantitative RT-PCR. The Full Spectrum RNA Amplification Kit from System Biosciences was designed to address this issue. Using proprietary amplification technology, the Full Spectrum method uniformly amplifies RNA from samples as small as 5 nanograms. In a single tube, in under three hours, it is possible to generate enough amplified template for hundreds of qRT-PCR reactions. The unique sequence independent priming method used in the Full Spectrum kit enables the analysis of the entire mRNA transcript, even in degraded mRNA samples. In comparison, standard T7/IVT amplification methods which use oligo-dT priming amplify only the extreme 3’ end of the transcript, making analysis of alternative splicing impossible. Aside from this 3’ end bias, T7/IVT methods take days to perform, require multiple purifications, and require high quality non-degraded RNA samples.
Full Spectrum RNA Amplification Maintains Relative Transcript Quantities
Your RNA amplification method must amplify all transcripts equally, so you have confidence that you’re measuring relevant biological expression levels. To demonstrate the uniformity of amplification achieved with the Full Spectrum RNA Amplification Kit, expression levels of 50 human genes were measured before and after amplification of 20 ng total RNA, and the Ct values were plotted in Chart 1A. The identical experiment was performed using a T7/IVT method with the exception that 50 ng total RNA was used, and the results were plotted in Chart 1B. As shown, the Full Spectrum method (R = 0.940) produces results that meet and exceed those of the T7/IVT method (R = 0.770). The data supports the assertion that the Full Spectrum method maintains relative transcript quantities during the amplification process.
Chart 1. Plot of Ct values for 50 genes obtained from non-amplified and amplified cDNA with the Full Spectrum method (1A) and T7 IVT method (1B).
Identify Differentially Expressed Genes with Full Spectrum RNA Amplification
To demonstrate the ability of the Full Spectrum method to identify differentially expressed genes by real time qPCR, we compared Ct values obtained from two different tissue sources. Of the 50 total genes we analyzed in Chart 1, TaqMan assays were prepared for 38. The first of these tissue sources was human universal RNA, which is a mixture of several tissues, and the second was kidney. Again, we compared these results with both amplified and non-amplified mRNAs and also compared mRNA amplified by the Full Spectrum method and the T7/IVT method. We then plotted the differences in Ct values by subtracting the Ct value obtained from universal RNA from the obtained kidney Ct (Charts 2A and 2B). As shown, the Full Spectrum RNA Amplification Kit identifies the same differentially expressed genes as the T7/IVT method, but with a much higher correlation coefficient.
Chart 2. Plot of ΔCt values. Kidney RNA Ct—universal RNA Ct from non-amplified and amplified cDNA with Full Spectrum (2A) and T7 IVT (2B) methods. Color coded dots represent the same gene on both plots.
Identification of Rare Alternative Splice Variants with Full Spectrum™ Transcriptome-Wide RNA Amplification
In genomic research today, there is an increased interest in identifying and quantifying of alternatively spliced transcripts. Now it appears that as many as 75% of the estimated 30,000 gene transcripts are alternatively spliced. Alternative splicing has been found to be important for various normal cellular activations such as development, differentiation, and programmed cell death. Alternative splicing has also been found associated with disease phenotypes such as hypertension, cancer, and even obesity. Identification and analysis of alternative splicing can be difficult for several reasons, among which are the following:
- Alternative splicing can occur all along a gene transcript. Both 5’ and 3’ ends of the mRNA sequence need to be preserved. This can be a problem if the gene transcript is very long or if secondary structure impedes reverse transcription.
- Some alternative splice variants are very low in abundance in many cells. Studies of low abundance transcripts require an amplification step, as has been done in the following UCLA study.
Experimental Determination of Splice Variants
A recent study was undertaken by UCLA scientists to experimentally verify splice variants predicted from EST database analysis to be tissue-specific and mouse strain-specific. An example of one such gene’s transcripts is presented below.
Fig 1. Genomic representation of splicing patterns for brain/testis-specific and mouse strain-specific transcripts (at left), and predicted transcript sizes by EST database analysis (at right).
The starting amount of total RNA for the unamplified RT reaction was 1 µg. First strand cDNA was primed with both oligo-dT and random hexamers. To amplify RNA, 0.5 µg was used in our Full Spectrum™ RNA Amplification kit protocol with 15 cycles of amplification. PCR products generated using forward and reverse primers (Fig. 1) were sequenced to verify alternative splicing.
With Full Spectrum™ RNA Amplification technology, you can:
- Identify and quantify alternative splicing in both long and rare gene transcripts
- Examine differential gene expression in clinical samples in which the RNA may be partially degraded
- Examine gene expression in small numbers of cells such as those obtained from laser capture microdissection.
- Amplify RNA with no purification steps, using fewer tubes, and with fewer pipetting steps using Full Spectrum’s 1-tube, 2-step, 3-hour protocol
Our Full Spectrum™ line of RNA Amplification products can help facilitate alternative splicing research:
Amplify Viral and Poly A- RNA with Full Spectrum Amplification of Viral RNA
Figure 1. Human coxsackievirus was spiked into cerebral spinal fluid (CSF) @ 10-4 TCID diluted with poliovirus RNA. Less than 50 ng of the RNA was amplified with the Full Spectrum Amplification Kit. Conditions of PCR were: 1° PCR, 20 cycles; 2° PCR, 38 cycles. 100bp size markers (M).
Amplification of Poly A- RNA
Figure 2. Human Histones H1a, lacking a poly A tail, and H1c, with a poly A tail, were examined from 50 ng mouse universal RNA. Control b-actin is shown amplified with 25 cycles, and histones with 28 cycles. RT-PCR primers were designed to amplify both the 5' and 3' ends of the mRNAs.
Kit Components
40 µl | Universal Primer Mixture |
10 µl | Reverse Transcriptase |
15 µl | Control RNA (25 ng/µl) |
| 30 µl | 5X Reverse Transcriptase Buffer |
| 20 µl | Dithiothreitol (DTT) |
50 µl | dNTP Mix |
| 1.2 ml | RNase-free Water |
| 150 µl | PCR Buffer |
25 µl | Full Spectrum PCR Primer |
25 µl | PCR Polymerase |
The kits are shipped in blue ice and should be stored at -20°C upon receipt. Properly stored kits are stable for 1 year from the date received.
