Although standard RNA-Seq has helped researchers obtain useful data from full transcripts, in the vast majority of cases scientists only need to perform differential gene expression analysis (DGE), which does not require information from the entire transcript. The 3’Tag RNA-Seq protocol was developed to lower costs and to help researchers focus on DGE data. This method generates DGE data with only one library molecule per transcript, which is complementary to the 3′-end sequence. Due to its selectivity, much less sequencing depth is required for 3’Tag RNA-Seq compared to standard RNA-Seq [1].

3’Tag RNA-Seq is commonly used for the analysis of gene expression in a variety of applications, including transcriptomics, epigenomics, and gene regulation studies.

3’Tag RNA-Seq vs. Standard RNA-Seq

3’Tag RNA-Seq is a protocol developed to obtain gene expression profiling data with a high signal-to-noise ratio at a low cost, and it differs from the classic RNA-Seq (polyA-selection) technique in a few key ways, even though both are based on targeting the eukaryotic messenger RNA (mRNA) molecules that have poly-A tails at their 3′ end.

In the standard RNA-Seq method, the extracted mRNAs are sheared into fragments and then reverse transcribed into complementary DNA (cDNA). Then this cDNA is sequenced. This fragmentation can sometimes introduce bias into the results, because the number of reads corresponding to each transcript is actually proportional to the number of cDNA fragments rather than the real number of transcripts. Longer transcripts can be sheared into more fragments, which means there may be more reads that correspond to them than the shorter transcripts. The results may falsely show that the genes with longer transcripts were expressed at higher levels than the genes with the shorter transcripts [2].

In 3’Tag RNA-Seq, the extracted mRNAs are actually not sheared into fragments at the beginning of the protocol. Instead, the cDNAs are only reverse transcribed from the 3′ end and only one copy of cDNA is generated for each transcript. Thus, when these cDNAs are sequenced, the number of reads directly reflects the number of transcripts of a gene, and there should be no bias for longer transcripts [2].

When is 3’Tag RNA-Seq recommended?

3’Tag RNA-Seq is recommended when you have eukaryotic samples and are only interested in DGE analysis for mRNA with polyA-tails. With this protocol, you will receive a very high-signal, low-noise gene expression profile at a much lower cost than standard RNA-Seq, and your samples don’t even have to be as high quality as many standard polyA-selection kits require. However, if you need any transcript-splicing information, have prokaryotic samples, or are interested in any types of RNA outside of mRNA with polyA-tails, then 3’Tag RNA-Seq is not recommended [3].

Here are some examples of articles where researchers used 3’Tag RNA-Seq:

• In this study [4], 3’Tag RNA-Seq was used to determine if the researchers’ treatments enhanced Natural Killer (NK) cell activation and expansion in dogs with cancer. The NK cells they targeted are cytotoxic immune cells capable of recognizing heterogenous cancer targets, which makes them very promising targets for use in cellular immunotherapy.
• The researchers here [5] performed 3’Tag RNA-Seq to identify the genes that were differentially expressed between healthy and injured rat tissue in order to determine which genes changed in response to injury.
• In this paper [6], 3’Tag RNA-Seq was used to determine the differential gene expression between bovine ovarian cortex cells treated with human-recombinant FSH or vehicle (control). The researchers wanted to study the effects of this folliculogenesis-promoting factor in preantral follicles, which serve as a reservoir of female gametes that can be used in assisted reproduction in humans and other animals.

How can Genohub help you?

Genohub and our 3’Tag RNA-Seq partners are knowledgeable in every step of the process, including extraction, library preparation, sequencing and data analysis. Regardless of your next-generation sequencing (NGS) experience, we will help define your project, present you with the best quote from our network, connect you directly to our NGS partner, manage the project while it’s progressing, see to it that the results are delivered to you in a secure data bucket, and then make sure all your needs were met before considering the project complete. All these steps will occur on the Genohub platform, and we will be here to support you every step of the way. 

Specifically, our 3’Tag RNA-Seq partners have experience extracting from many different types of tissues and cell types, but they can work just as well with your RNA samples or libraries that you have prepared yourself. Our partners can also sequence at their most cost-effective configuration and then trim the reads down to what you need for your specific 3’Tag RNA-Seq analysis. 

We know that each research project is unique, so we are open to working with your custom analysis needs! Get started today by letting us know about your 3’Tag RNA sequencing project here: https://genohub.com/ngs/ .

References

1. UC Davis DNA Technologies Core. “When do you recommend 3′-Tag RNA-seq?” DNA Tech Genome Center UC Davis, 24 August 2023, https://dnatech.genomecenter.ucdavis.edu/faqs/when-do-you-recommend-3-tag-rna-seq/.
2. Ma, F., Fuqua, B.K., Hasin, Y. et al. A comparison between whole transcript and 3’ RNA sequencing methods using Kapa and Lexogen library preparation methods. BMC Genomics 20, 9 (2019). https://doi.org/10.1186/s12864-018-5393-3
3. UC Davis DNA Technologies Core. “Gene Expression Profiling with 3′ Tag-Seq.” DNA Tech Genome Center UC Davis, 24 August 2023, https://dnatech.genomecenter.ucdavis.edu/tag-seq-gene-expression-profiling/.
4. Razmara A, Farley L, Harris R, et al 272 Pre-clinical evaluation and first-in-dog clinical trials of intravenous infusion of PBMC-expanded adoptive NK cell therapy in dogs with cancer Journal for ImmunoTherapy of Cancer 2022;10:doi: 10.1136/jitc-2022-SITC2022.0272
5. Danielle Steffen, Michael J. Mienaltowski, Keith Baar, Scleraxis and collagen I expression increase following pilot isometric loading experiments in a rodent model of patellar tendinopathy, Matrix Biology, Volume 109, 2022, Pages 34-48, ISSN 0945-053X, https://doi.org/10.1016/j.matbio.2022.03.006.
6. Candelaria J., Rabaglino B., Denicol A. (2020) 125 Transcriptomic changes in bovine ovarian cortex in response to FSH signaling. Reproduction, Fertility and Development 32, 189-189. https://doi.org/10.1071/RDv32n2Ab125