Expression levels of functional mRNA denote the essential functions that are required to retain life under various conditions. Thus, the degree of mRNA expression is widely employed to understand the cellular conditions (Wang et al. 2009; Qin et al. 2016). Therefore, northern blotting, RT-qPCR, and DNA microarray were employed to quantify mRNA. Furthermore, RNA sequencing using a next-generation DNA sequencer and Digital RT-PCR is essential to study “omics”. Technologies focusing on mRNA are steadily developing and offer higher sensitivity, whereas techniques for determining the quality of extracted mRNA are still not at par. To analyze functional mRNA, it is essential to establish a more precise RNA quality control method (Kashofer et al. 2013; Li et al. 2015).
In this study, we validated a new RNA quality control method using an external standard. We used the RNA Solutions by Qualitative Analysis (AIST, Japan) as the external standard RNA. Standard RNAs have the potential to be used to evaluate mRNA directly because standard RNAs are designed based on human mRNA (Tong et al. 2006). Standard RNA has already been used to evaluate RNA yield (Takahashi et al. 2013). However, in this study we focused not only on yield but also RNA degradation. Standard RNAs are available in five different types, viz., three each of 533-nt and two each of 1033-nt, and are designed in a way that they share low homology sequences with natural sequences. Furthermore, they have the potential to be used to directly evaluate certain factors simultaneously. For this study, we evaluated the yield of mRNA from cells, inhibition by contaminants, i.e. unknown cellular component such as DNA polymerase, and degradation of mRNA by using standard RNA.
RNA yield is a factor denoting the quality, but it is usually ignored as a base for evaluation of quality control. Low yield of RNA suggest that the extraction procedure is not appropriate or that cellular disintegration causes RNA damage (Kashofer et al. 2013). We evaluated the final yield of total RNA and not the efficiency of RNA extraction. Comparison of RNA expression from different samples with different efficiencies of RNA yield may affect the final outcome. Therefore, we evaluated the final yield of RNA and the efficiency of yield during RNA extraction. In this study, RNA was extracted from
The prepared RNA samples are not always free of contaminants since they can be contaminated with proteins and polysaccharides; these contaminants can lead to unwanted enzymatic reactions that inhibit RNA extraction and, hence, denote false positive results (Pionzio and McCord 2014). A260/A230 and A260/A280 ratios are generally used as a base for determination of RNA quality (Sambrook et al. 1989; Manchester 1996). Absorbance at 230 nm usually denotes contamination by organic solvents or TE. Absorbance at 260 nm denotes mass of nucleic acids; RNA and DNA. Absorbance at 280 nm denotes contamination by proteins or DDT. Contamination, measured by the A260/A280 ratio, is considered to inhibit reaction of enzymes (e.g., reverse transcriptase and DNA polymerase). A260/A280 and A260/A230 ratios are widely accepted for evaluating the contamination in the prepared RNA samples. However, this ratio does not directly evaluate inhibition. Thus, standard RNA can be used to evaluate the degree of unknown contamination that may inhibit enzyme reaction. In this study, we employed real-time qPCR to quantify the final value of standard RNA and to determine the quantity of inhibition in RNA-extraction solutions of
Evaluation of mRNA degradation is not easily accepted by organizations that are responsible to record the values obtained from all prepared mRNA samples. RNA Integrity Number (RIN) value and 18S/28S ribosomal ratio are the most used parameters for determining mRNA quality control (Imbeaud et al. 2005). This method considers two kinds of rRNA as internal standards. The extracted total RNA is subjected to capillary gel electrophoresis, and an electropherogram is constructed from the fluorescence for determining retention time. By comparing two peaks of rRNA and with the ideal peak, RNA quality is evaluated. However, because this method evaluates rRNA that comprises a major portion of the total RNA, it does not always reflect the quality of mRNA, which comprises a small portion of the total RNA (Feng et al. 2015). While standard RNA is designed on the basis of mRNA, it has the potential to evaluate mRNA quality. In this study, we evaluated mRNA degradation using standard RNA. We also validated the difference in degradation of RNA structure from the 3’ and 5’ ends.
This study, hence, helps in establishing a method using external standard RNA that can be used to directly evaluate the extracted mRNA quality.
Primers used in the RT-qPCR.
Target | Forward Primer | Reverse Primer |
---|---|---|
1000-A | 5’-CAACCGGTGTGATCAGGACA-3’ | 5’-AGGACAGTCCGCATAAGCAC-3’ |
1000-B | 5’-TACCAGCGCTTCTGTACGAC-3’ | 5’-GAGCTGTATCCGTGCCGTAA-3’ |
500-A | 5’-TCGCAGGCCTAATACGTGTC-3’ | 5’-CGTGAATCTCGGAGCGGTAA-3’ |
500-B 3’end | 5’-GGGTAGCGATTTAACGACTCG-3’ | 5’-CAGAGCCTGCCTTATCGTGA-3’ |
500-B middle | 5’-CCGAACGCTACGTGACGATA-3’ | 5’-ATCTACATGTTCCGTGCGCA-3’ |
500-B 5’end | 5’-AGACTAAATCTCGGCGTCGG-3’ | 5’-TAGATAGGGTCCGCATGACG-3’ |
500-C 3’end | 5’-GCACGACCGAATTATGCACC-3’ | 5’-AACCACTGACGTGAGCGATT-3’ |
500-C middle | 5’-TAGACGCGCCTTACTCCTCT-3’ | 5’-TAGTGGAGCTCGCGGATTTG-3’ |
500-C 5’end | 5’-GGACTAAACGCACTGAATACCG-3’ | 5’-ATCGCCCGTACTATCCGGTA-3’ |
Inhibitory effect (%) = 100 − [(A − B) ÷ C] × 100,
where A denotes the concentration of standard RNA in the RNA-extracted solution in RT-qPCR, B denotes the concentration of standard RNA in the RNA-extracted solution, and C denotes the concentration of the added standard RNA.
Industrially available standard mRNA was used for quality control of prepared total RNA from
Before RNA extraction, standard RNA of known concentration was added into the sample tubes. We evaluated the mRNA yield, by tracing the amount of added standard RNA during the experiment. We added standard RNA during all key steps in the protocol. Total RNA was extracted from
Fig. 1 shows the yield of standard RNA in each RNA extraction step. In RNA extraction from
To evaluate the inhibitory effect caused by contaminants, we re-added standard RNA at the same concentrations to the extracted RNA solutions and measured. Table II shows the inhibitory effect of RNA extract solution from
Inhibition of RT-qPCR by RNA extract solutions from
Species | 1000-A | 1000-B | 500-A |
---|---|---|---|
–1.1 ± 4.8 | 50.4 ± 4.0 | 0.3 ± 3.1 | |
7.0 ± 3.2 | 44.6 ± 3.0 | 27.7 ± 2.8 |
We evaluated RNA degradation using standard RNAs. Standard RNA 500-B and 500-C were degraded by
Fig. 2 shows the survival rate of each region on both standard RNAs over time. We discovered that RNA degradation by
We attempted to evaluate the standard RNA quality of samples degraded by
Survival of 3’ end and 5’ end and the ratio of 3’ end to 5’ end regions of standard RNAs after degradation with
Standard RNA | 500-B | 500-C | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
Degradation time (min) | 0 | 10 | 20 | 60 | 180 | 0 | 10 | 20 | 60 | 180 |
3’ end (survival %) | 100 | 73.5 | 45.2 | 32.1 | 6.3 | 100 | 76 | 59.2 | 26.7 | 3.2 |
5’ end (survival %) | 100 | 85.9 | 71.2 | 67.9 | 6.4 | 100 | 88.9 | 92.9 | 67.2 | 8.6 |
3’ end /5’ end (ratio) | 1 | 0.86 | 0.63 | 0.47 | 0.99 | 1 | 0.86 | 0.64 | 0.4 | 0.37 |
We demonstrated that RNA can be easier degraded at the 3’ end than the 5’ end by
We calculated the yield of mRNA in each step for both
This information could fast-track the development of advanced preparation procedures. The use of standard RNA has the potential to evaluate and normalize the differences in yield of RNA from different organisms and to elucidate the reason behind low yields from specific organisms. The mRNA yield is usually not considered for determining the quality as the yield can be easily increased by increasing the volume of biological sample used to extract mRNA. Biological resources are often limited, and clinical samples and the samples obtained from the crime site are often in minute quantities, so the cost for performing forensic analysis is not economical (Georgiadis et al. 2015). Intestinal jejunum and skin are representative organs that present difficulty in mRNA extraction (Berglund et al. 2007; Heumüller-Klug et al. 2015). Thus, standard RNA may contribute toward advancing the quality of mRNA yield from these organs.
The extracted RNA may contain impurities that cause inhibitory effect on enzymatic reactions, leading to false positive results (Kashofer et al. 2013). We attempted to evaluate inhibition for real-time qPCR using standard RNA but result was incorrect. The possible reason for this result may be that the sequences of standard RNA were affected by RT-qPCR. Standard RNA is an external standard, and because the exact concentration is known, we may directly evaluate inhibitory effect. The direct evaluation of inhibitory effect disagrees with the lack of manual skill in this experiment. Furthermore, it should be noted that standard RNA may exert an inhibitory effect by interaction with secondary structures and other nucleic acids (Bustin et al. 2009).
We evaluated RNA degradation using standard RNAs. Standard RNAs were degraded by
To confirm one-dimensional degradation and to evaluate RNA degradation, standard RNAs are suitable. In conclusion, we propose the following quality control method for RNA degradation: first, standard RNA must be added during the procedures of RNA isolation; secondly, RNA degradation must be evaluated by comparing the structure and the ratio of the 5’ end and 3’ end of standard RNA. Furthermore, the quality of prepared RNA may be evaluated by measuring the 3’ end of the standard RNA as it is more prone to degradation than the 5’ end. Hence, RNA degradation quality control can be evaluated by comparing the 5’ end and the 3’ end of standard RNA for samples that exhibit biased RNA degradation. Further studies are required in other types of cells, including blood cells, visceral cells, and cultured cells.