Sample Submission Guidelines
Total RNA Sequencing Q&A
- General Questions
- What kinds of Total RNA Sequencing Services do you provide?
- CD Genomics Total RNA Sequencing service is a powerful and comprehensive solution for analyzing the cellular transcriptome. Whole RNA-Seq has a wide range of applications, from basic studies of cell structure and function to the detection and analysis of various diseases. For example, changes in gene expression before and after a therapeutic intervention can be compared to determine the presence or absence of disease. RNA-Seq can also be used to detect alternative splicing, post-transcriptional modifications, and exon-intron analysis. The data obtained can provide valuable information on underlying cellular mechanisms, genome structure, and disease-inducing effects.
- How does Total RNA-Seq work?
- The RNA-Seq process revolves around the construction of a complementary DNA (cDNA) library for sequencing. Library construction begins with isolation of cellular RNA, followed by quality control assays to determine RNA integrity. Subsequently, target RNA in the library can be enriched using either removal or a screening assay. RNA is then reverse transcribed into cDNA prior to sequencing.
- Should I choose total RNA sequencing or mRNA sequencing?
- Whether to use total RNA-Seq or mRNA-Seq depends on the purpose of the experiment. Total RNA sequencing provides the most comprehensive analysis of the entire transcriptome. If eukaryotes are studied and only coding regions are of interest and limited starting material is available, mRNA sequencing is the ideal solution.
- What kinds of RNA species can be obtained by total RNA sequencing?
- Total RNA-Seq is the sequencing of RNA that has had ribosomal RNA (rRNA) removed, which contains a variety of RNA molecules. These include precursor messenger RNA (pre-mRNA), messenger RNA (mRNA), and many types of non-coding RNA (ncRNA) such as transfer RNA (tRNA), microRNA (miRNA), and long non-coding RNA (lncRNA, transcripts longer than 200 nucleotides that have not been translated into proteins).
- Which strategies to use to improve RNA sequencing data quality?
- Either ribosome removal or mRNA enrichment can improve the quality of sequencing data. Both approaches enable sequencing of only the target RNA molecule and minimize the waste of sequencing reads.
Removal of rRNA transcripts allows more sequencing reads to be focused on the desired transcripts, thus improving the sensitivity of sequencing. The rRNA removal step is particularly important if the expression level of the desired transcript is quite low. Poly-A selection is sufficient to study mRNA in eukaryotes. And analysis of lncRNA or bacterial transcripts requires depletion of rRNA.
- Either ribosome removal or mRNA enrichment can improve the quality of sequencing data. Both approaches enable sequencing of only the target RNA molecule and minimize the waste of sequencing reads.
- What sequencing platforms are used for RNA-Seq?
- Standard, strand-specific, single-cell, small and ultra-low input RNA-Seq uses short-read sequencing on Illumina platforms such as NovaSeq and HiSeq.
- Can you analyze small RNAs or miRNAs?
- Yes, total RNA-Seq provides information about coding RNAs as well as non-coding RNAs such as lncRNAs and miRNAs
- How much sequencing data do I need in Total RNA Sequencing?
- Total RNA-Seq requires more sequencing data (typically 100-200 million sequencing reads per sample). For mature species such as human, rat, and mouse, 6G bases are sufficient to study only mRNA expression, and 12-15G is recommended for lncRNA expression. 5-10M reads are basically sufficient for small RNA sequencing. For other species or needs, you can contact us for more information.
- What bioinformatic analysis does total RNA sequencing provide?
- We have a complete bioinformatics support team to provide additional data analysis for routine and custom requests. This includes, but is not limited to, alternative splicing analysis, identification of novel transcripts, fusion gene analysis, SNP detection, as well as differential analysis, trend analysis, and enrichment analysis.
Visit the following pages for more information about the Bioinformatics Analysis of RNA-Seq.
- We have a complete bioinformatics support team to provide additional data analysis for routine and custom requests. This includes, but is not limited to, alternative splicing analysis, identification of novel transcripts, fusion gene analysis, SNP detection, as well as differential analysis, trend analysis, and enrichment analysis.
- Sample Preparation
- What sample types do we support?
- We support a wide variety of samples such as cells, tissues, FFPE, blood, urine, and exosomes. For more information, please read our specific sample submission or submit an inquiry here.
- How to extract total RNA?
- RNA extraction starts with the homogenization of the sample followed by lysis of the cells to release the RNA. RNA is then conditioned to bind to a capture column, and washed to remove salts and other contaminants before it is eluted. Any contaminating genomic DNA can be removed with column-based or enzyme-based methods.
Check out our Total RNA Extraction/Purification Protocol for more information.
- RNA extraction starts with the homogenization of the sample followed by lysis of the cells to release the RNA. RNA is then conditioned to bind to a capture column, and washed to remove salts and other contaminants before it is eluted. Any contaminating genomic DNA can be removed with column-based or enzyme-based methods.
- How should I take samples?
- RNA is very degradable and preference is given to fresh, vigorously growing tissues. Cell samples need to be at least 107. whole blood samples should be no less than 5 ml. Samples need to be frozen in liquid nitrogen and stored at -80°C. Samples need to be securely sealed and shipped on dry ice.
- How to Order
- How should I prepare and send samples?
- Review our sample delivery guidelines for instructions on preparing and sending samples for total RNA sequencing.
- What kind of data will I receive?
- Our data delivery and format vary by platform/application and follow the NGS industry standards. Sequencing data, filtered and unfiltered, is typically delivered as fastq file. Reference alignments are provided as .bam files. SNP calls and/or variants are provided as VCF files. We also offer custom data formats and reports to meet your specific requirements.
For research purposes only, not intended for clinical diagnosis, treatment, or individual health assessments.
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