Relative Quantification with the LightCycler® 480 Real-Time PCR System
Featured Study: Evaluation of RNA stabilization treatments prior to RNA isolation from lung biopsies
Respiratory Genetics Group, Divisions of Infection, Inflammation & Repair and Human Genetics, School of Medicine, University of Southampton, United Kingdom
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Quantitative real-time PCR is an established laboratory technique for assaying mRNA transcript levels. There are several tissue processing steps prior to PCR that can affect the accuracy and precision of real-time-PCR based mRNA transcript quantification [1, 2]. RNA molecules can be degraded, so the researcher has to take steps to prevent RNase contamination. Liquid-based stabilization reagents are available commercially and preserve RNA integrity by inhibiting RNase activity in tissue prior to isolation. It is important to evaluate the effectiveness of any RNA stabilization treatment in a new experiment before the quantification results can be reliably interpreted.
The aim of the research project was to determine the effectiveness of a stabilization reagent in maintaining RNA integrity in rat lung tissue, prior to isolation. The RNA content in three rat lung samples was stabilized either by submersion in a commercial stabilization reagent (reagent A) or by the traditional liquid nitrogen freezer-fracture method. Intra- and inter-sample variability between the two methods was determined by relative quantification of adam33 RNA transcripts against Actb RNA transcripts.
Materials and Methods
Two 100 mg biopsies were taken from three rat lung samples. The first set of biopsies was treated with stabilization reagent A for 24 hours at -20°C prior to isolation, according to manufacturer’s instructions. The second set of biopsies was snap-frozen in liquid nitrogen and fractured before being stored in 1 ml of the isolation solution for 24 hours at -20°C. All samples were homogenized using ceramic beads before isolating the RNA. 1 µg of RNA was reverse-transcribed and the relative quantification of adam33 (GeneID: 110751) and Actb (GeneID: 81822) mRNA was determined by Universal ProbeLibrary probe assays on the LightCycler® 480 System.
Results and Discussion
Actb amplification in the freeze-fracture treated samples occurred on average 5.38 Cp values earlier than in the stabilization-reagent-A treated samples (see Figure 1 and Table 1). The adam33 relative-quantification-normalized ratios differed by up to 8.8-fold in concentration when comparing biopsies from the same lung (see Figure 2 and Table 1), and on average there was a 4.51-fold difference in mean normalized ratios between the two treatments (see Table 2). The inter-sample variability is less in the freeze-fracture treated samples (1.05 ± 0.158, mean normalized ratio ± std error, with 26% CV) than in the stabilization–reagent-A treated cDNA template (4.75 ± 1.37 with 51 %CV).
Figure 1: Amplification Curves of adam33 and Actb Genes. Blue plots = freeze-fracture treatment, Green plots = Stabilization reagent A treatment, Red plots = Standard curve analysis, Black plots = negative control reactions. Y-axis is the change in fluorescence and x-axis is the PCR cycle number (Cp value). The freeze-fracture treated samples are consistently amplifying earlier than the stabilization reagent A treated samples for both the Actb (left panel) and adam33 (right panel) genes. PCR efficiencies estimated from the slope of the standard curves were 1.96 (error = 0.009) and 1.99 (error = 0.013) for adam33 and Actb, respectively.
Figure 2: Relative Quantification Results Chart. Light blue bars are the calibrator-normalized concentration values used to determine the relative quantification of adam33 to Actb. Y-axis is the log10 concentration ratios and each result set is shown on the x-axis. It is clear to see the small change in relative concentration values of adam33 in the freeze-fracture treated lung biopsy group compared to the stabilization-reagent-A treated samples.
|Sample Name||Treatment||Sample Type||Median adam33 Cp values||Median Actb Cp values||Conc. Ratio||Conc. Ratio STD||Norm. Ratio||Norm. Ratio STD|
|1||Stabilization Reagent A||Unknown||28.37||22.22||1.00E-02||2.53E-03||2.26||0.46|
Table 1: Relative quantification results.
|Treatment||Mean Normalized Ratio||Mean Fold Difference|
|Stabilization Reagent A||4.75|
Table 2: Mean normalized ratios of adam33 between treatments.
In our hands, the freeze-fracture method is superior as it consistently maintains the integrity of RNA in rat lung tissue samples prior to isolation. The results from this study show that a large proportion of the variability between biological replicates could be attributed to an ineffective stabilization treatment. While the liquid nitrogen freeze-fracture method is time-consuming when processing numerous tissue samples, utilization of this treatment assures us that our qPCR results are reliable.
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