Small RNA Sequencing Q&A

  • General Questions

  • Can small RNA sequencing be done if there is no reference genome for the species?
    • Small RNA sequencing can be performed for species without a reference genome, but it is necessary to do the splicing of transcripts without reference transcriptome first and use the transcripts as reference sequences for small RNA sequencing analysis.
  • For species without reference genome, how to perform small RNA sequencing for target gene prediction?
    • We suggest you to do transcriptome sequencing to discover large-scale transcripts, construct Unigene library of the target species. Then, based on this, small RNA sequencing is performed to get known small RNAs and predicted new miRNAs through annotation, and target gene prediction is performed by comparing with genes in the Unigene library, and finally, functional annotation of target genes is completed.
  • How to perform quality control of Small RNA sequencing libraries?
    • The library construction method for Small RNA sequencing using Illumina Hiseq2000 is as follows:
      (1) PAGE gel purification of small RNA molecules of specific size.
      (2) 5' splice ligation and purification.
      (3) 3' splice ligation purification.
      (4) RT-PCR amplification.
      (5) Small RNA library purification.
  • Can I use the results of small RNA sequencing to analyze tRNA?
    • No, you cannot. Transfer RNA (tRNA) is universally expressed non-coding RNA with a length of 70-90 nucleotides (nt). tRNA has a stable tertiary structure and a high degree of base modification, methylation, translocation reactions within nucleosides, and reduction reactions (necessary for tRNA function).
  • Can I use total RNA to prepare a Small RNA library? Or do I have to isolate or enrich Small RNA as a starting sample?
    • Small RNA libraries can be prepared using total RNA. It is not necessary to enrich Small RNA if the concentration of Small RNA is higher than 0.5%, and it is recommended to use total RNA with a RIN value greater than 7. You can contact us for more information about our tRNA sequencing service.
  • Can I use enriched Small RNA to prepare a Small RNA library?
    • Yes, you can use enriched Small RNA, and if the amount of enriched Small RNA is close to 10 ng, dilute the junction at the recommended dilution rate of 100 ng of total RNA as described in the instructions. If the amount of enriched Small RNA is close to 100 ng, dilute the junction at the recommended dilution rate of 1 ug of total RNA as described in the instructions.
  • How to perform experimental validation after sRNA sequencing?
    • There are a number of methods for subsequent experimental validation of sRNA.
      1. qPCR, which verifies microRNA expression.
      2. RIP-seq, which enriches RNA with methylation modification or RNA that binds specifically to the target protein by RIP technology. High-throughput sequencing of the enriched RNA is performed and compared with the genome to obtain information on the location of microRNAs that bind specifically to the target protein, etc.
      3. Luciferase reporter system.
      4. Construction of overexpression vectors or knockdown of target genes.
  • What are the features of our Small RNA sequencing service?
    • (1) Small RNA molecules can be studied directly at the nucleotide level, without the problems of cross-reactivity and background noise caused by the fluorescent mimic signals of traditional microarray hybridization, facilitating the differentiation of different Small RNA molecules of the same family as well as similar sequences.
      (2) Enables high-throughput analysis of species without the need for prior sequence information as well as secondary structure information.
      (3) High sensitivity and high sequencing throughput provide data depth and coverage for the discovery and study of Small RNA molecules and enable the detection of rare transcripts with low abundance.
      (4) The raw data generated by sequencing is compatible with a variety of analysis software, allowing the annotation of genomic information of Small RNAs and analysis of their expression levels, the ability to annotate known Small RNAs using the Small RNA database, and the ability to further analyze unmatched data to discover new Small RNA species and isoforms and find information for more in-depth studies.
  • How to perform raw signal analysis of sRNA sequencing
    • (1) Data quality control: The quality of sequencing data is an important prerequisite for the reliability of information analysis results. sRNA raw data need to go through quality control processes such as junction sequences, removing N-containing base sequences, removing low quality base sequences, length screening, etc. to obtain high quality sequencing data, and count the number of small RNA species and length distribution.
      (2) Reference genome alignment: Starting from the clean reads of each sample after length screening, the reads are aligned to the reference genome and the alignment results are counted.
      (3) Known miRNA analysis: reads are compared with miRNA database to identify known micro RNAs.
      (4) Non-coding RNA analysis: compare reads with ncRNA sequences of species or Rfam database to identify rRNA, tRNA, snRNA, snoRNA.
      (5) Novel miRNA prediction: perform novel miRNA prediction using miREvo and perform analysis such as base preference and secondary structure.
      (6) Gene expression and differential analysis: quantify known miRNAs and new miRNAs based on the comparison results, and perform differential miRNA analysis to obtain differentially expressed miRNAs between samples or subgroups.
      (7) miRNA target gene prediction: perform differential miRNA target gene prediction using both miRanda and RNAhybrid software.
      (8) Functional enrichment: Based on the target genes of differential miRNAs between samples or subgroups, we obtain significantly enriched functional information by functional annotation databases, such as GO, KEGG, etc.
  • If there is no genome sequence, how to find the relevant miRNAs and their target genes?
    • The first way is to refer to the existing literature, for example, miR156, miR172 has a role in nutritional growth to reproductive growth. If genome sequencing is not possible, then transcriptome sequencing and small RNA sequencing should be performed to analyze the transcriptome as a reference. Finally, the sequences can be submitted directly to some online software such as psRNATarget for target site prediction.
  • When performing sRNA sequencing, what relevant information do I need to provide in addition to the sample?
    • The genome of the respective species and the relevant exon, intron, repeat information is required. If the genome of the species is not available, information about the closely related species is required.
  • What is the Read/Tag length for Small RNA sequencing using Illumina Hiseq2000? What is the recommended coverage depth?
    • For Small RNA sequencing, customers can choose the length of Small RNA they are interested in, which is 18-30 nt. For Illumina Hiseq2000, we recommend a sequencing length of 35 bp, after which the sequence information is trimmed to remove the splice sequence and leave only the Small RNA sequence. For Small RNA discovery and analysis studies, we do not recommend the DepthofCoverage during sequencing because of the need to perform expression analysis of Small RNA. Generally, at least 5 million reads are acquired per sample, which is enough information to ensure accurate sequence sequencing.
  • What factors affect Small RNA sequencing?
    • The influencing factors of sRNA sequencing are mainly as follows
      (1) The quality of the sample provided by the customer. In the process of Small RNA sequencing, sRNA needs to be isolated, and the quality and purity of the isolated Small RNA directly affect the sequencing results. Since RNA is easily degraded, the operations of RNA extraction and purification must be performed strictly according to the experimental requirements to ensure the quality of the samples.
      (2) Quality of library construction: Library construction requires PAGE gel to separate and purify Small RNA, and then connect the junction for purification before RT-PCR, in this process, careful operation is required to ensure that Small RNA is not degraded, and the purification process should ensure that the junction is removed cleanly, and the residual junction will have an impact on subsequent sequencing.
      (3) Control of the loading volume of sequencing library: This factor also affects the density of cluster generation to a great extent. Since the loading volume is very small, only 1-8 pg, the ability to accurately quantify microsamples also becomes an important factor affecting the sequencing throughput.

  • Sample Preparation

  • What types of samples can be processed by our sRNA sequencing service?
  • What kind of sample requirements are there for Small RNA sequencing?
    • (1) Sample purity requirement: OD value should be between 1.8 and 2.2; 28S:18S is at least 1.5 for electrophoresis detection.
      (2) Sample concentration: total RNA concentration should not be less than 200 ng/μL, please do not use over-column method to extract total RNA, total sample should not be less than 20 μg (Small RNA: total RNA > 0.3%). Or provide Small RNA samples with concentration greater than 20ng/μL and total amount greater than 1 μg.
      (3) Small RNA samples should be stored at -20°C; please provide the specific concentration, volume, preparation time, solvent name and species origin of Small RNA samples. Please also include QC data including electrophoresis gel chart, spectrophotometric or Nanodrop instrument detection data. If multiple sample preparation is required, please provide the samples required for multiple sample preparation.
      (4) Sample transport: Samples should be transported in 1.5 ml tubes with the sample name, concentration and preparation time indicated on the tube and sealed with Parafilm. It is recommended to use dry ice for shipping and to use a faster shipping method to reduce the possibility of sample degradation during shipping.
  • What are the special requirements for sample extraction for small RNA sequencing?
    • It is recommended not to use Qiagen and other companies' over-column kits or LiCl precipitation to avoid losing small fragments of RNA. if small RNA samples are provided directly, special kits for small RNA extraction can be used for extraction.
  • Why are there degraded rRNA sequences in the results?
    • Since total RNA is often slightly degraded and there is a natural degradation process in organisms, the data will contain a small percentage of degraded mRNA fragments. However, this percentage is usually very low and depends on the quality of the sample total RNA.
For research purposes only, not intended for clinical diagnosis, treatment, or individual health assessments.
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