For you to obtain an accurate picture of transcriptomes, you have to consider all the RNA transcripts in an organism regardless of whether it is a coding or non-coding.
Again, you need to isolate the entire RNA of an organism. For you to achieve this, instead of using fractions such as messenger RNA, you can opt for total RNA. This includes all the RNAs in a cell and other different RNAs and not just the messenger RNA or mRNA. Scientists are now delving deeper into RNA biology and transcriptomes. With this technique, they can measure gene and transcript abundance with ease while identifying known novel features of a transcriptome. Most total RNAs are obtained from various cells and tissues through a methodology called modified guanidine thiocyanate.
What makes total RNA desirable?
Even if you are working on low-quality or normal samples, total RNA-sequencing provides you with optimum coverage. It means you can be confident of the functionality of your genomics results. This follows the modern understanding of the complexity of transcription of RNA. Similarly, the functions of RNA has changed tremendously. This is why you have to consider all the RNA species available in an organism or tissue.
The use of ideal protocol or kit for each sample helps to ensure full recovery of top quality RNA which is great for representing low-copy sequences in your study. You can get thousands of these samples obtained from different human tissues including normal, tumor or tissues exhibiting other diseases.
By using the already obtained samples of total RNA, you can save your time and energy for your research. The hassles of locating and extracting RNA from different samples can be time-consuming. Most of them are ready-to-use for cDNA synthesis, Northern blotting, RNase protection analysis, qrt-PCR, microRNA study and RNA differential display among others. Additionally, it also allows you to identify all the biomarkers across a wide dynamic range of transcripts.
You can also get a full grasp of all the phenotypes of your interest in your research. You can also do your profiling with ease. Again, since abundant RNA species are removed, it leaves the transcripts of interest intact and fragmented thus allowing maximum library capture.
Common features of total RNA
- It passes through extensive quality control processes to ensure that they are of the highest-quality. To confirm the integrity of RNA, it is examined in a visual inspection to check for the intactness and presence of 18s and 28s ribosome RNA. A spectrophotometer may also be used to check quality and purity. It is also treated with DNAse I.
- They are isolated from various tissues some of which are hard to obtain;
- Polysaccharide decontamination;
- There is a high-efficiency reverse transcription;
- There is proper documentation of tissues’ clinical record.
Areas of application
Due to its desirability, total RNA has been utilized in various sectors in the research of genes and other clinical studies. The technique is valuable if you want to:
- To analyze splicing and alternate splicing tools;
- If you want to obtain the full genome-wide view of the entire transcriptome;
- Dig deep in your research for transcriptomes of human and non-human origin;
- Northern blotting, RACE and RT-PCR;
- cDNA library construction and synthesis;
- cDNA investigation ideal for profiling the study of gene expression;
- RNA primer extension and protection;
- mRNA purification procedures;
- The study of microRNA;
- Display of RNA and more.
How is it different from other sequencing strategies?
This methodology allows for both coding and non-coding RNA sequencing making it distinct from other transcriptome sequencing methods. Instead of undergoing the polyA+ selection, this technique subjects the entire RNA to rRNA depletion. This is because the rRNA makes the biggest percentage of the whole RNA.
The process of reducing rRNA is vital in allocating more additional sequencing reads to the transcripts of your interest. You can use it in a case where you want to analyze all the RNA transcripts of an organism. It enables you to explore all the available RNA species, unlike mRNA that allows you analyze specific coding part of a genome.
You can discover several new variants and alternative splicing in any genome of interest. It also allows you to understand the variants that affect the classification and progression of tumors. Furthermore, if you are working with organisms that have large and repetitive genomes like plants, this technique makes it easy for you to sequence them.