Editing Gene expression (section)

===mRNA quantification===
Levels of mRNA can be quantitatively measured by [[northern blotting]], which provides size and sequence information about the mRNA molecules.<ref>{{cite journal | vauthors = Ahmad W, Gull B, Baby J, Mustafa F | title = A Comprehensive Analysis of Northern versus Liquid Hybridization Assays for mRNAs, Small RNAs, and miRNAs Using a Non-Radiolabeled Approach | journal = Current Issues in Molecular Biology | volume = 43 | issue = 2 | pages = 457–484 | date = June 2021 | pmid = 34206608 | pmc = 8929067 | doi = 10.3390/cimb43020036 | doi-access = free }}</ref> A sample of RNA is separated on an [[agarose gel]] and hybridized to a radioactively labeled RNA probe that is complementary to the target sequence.<ref>{{Cite book | vauthors = Hoy MA |url=https://www.sciencedirect.com/book/9780124158740/insect-molecular-genetics |title=Insect Molecular Genetics |publisher=[[Academic Press]] |year=2013 |isbn=978-0-12-415874-0 |edition=3rd |pages=215–249 |language=en |chapter=Chapter 6 - Some Additional Tools for the Molecular Biologist |doi=10.1016/C2011-0-69839-4 |chapter-url=https://www.sciencedirect.com/science/article/abs/pii/B9780124158740000068}}</ref> The radiolabeled RNA is then detected by an [[autoradiograph]].<ref>{{Cite book | vauthors = Petrov A, Tsa A, Puglisi JD |title=Laboratory Methods in Enzymology: RNA |date=2013 |chapter=Analysis of RNA by analytical polyacrylamide gel electrophoresis |chapter-url=https://pubmed.ncbi.nlm.nih.gov/24034328/ |volume=530 |pages=301–313 |doi=10.1016/B978-0-12-420037-1.00016-6 |issn=1557-7988 |pmid=24034328|isbn=978-0-12-420037-1 }}</ref> Because the use of radioactive reagents makes the procedure time-consuming and potentially dangerous, alternative labeling and detection methods, such as digoxigenin and biotin chemistries, have been developed.<ref>{{cite journal | vauthors = Boyle A, Perry-O'Keefe H | title = Labeling and colorimetric detection of nonisotopic probes | journal = Current Protocols in Molecular Biology | volume = 3 | issue = Supplement 20 | pages = Unit3.18 | date = May 2001 | pmid = 18265226 | doi = 10.1002/0471142727.mb0318s20 }}</ref> Perceived disadvantages of Northern blotting are that large quantities of RNA are required and that quantification may not be completely accurate, as it involves measuring band strength in an image of a gel.<ref>{{cite journal | vauthors = Kuang J, Yan X, Genders AJ, Granata C, Bishop DJ | title = An overview of technical considerations when using quantitative real-time PCR analysis of gene expression in human exercise research | journal = PLOS ONE | volume = 13 | issue = 5 | pages = e0196438 | date = 2018-05-10 | pmid = 29746477 | pmc = 5944930 | doi = 10.1371/journal.pone.0196438 | doi-access = free | bibcode = 2018PLoSO..1396438K }}</ref> On the other hand, the additional mRNA size information from the Northern blot allows the discrimination of alternately spliced transcripts.<ref>{{cite journal | vauthors = Yang T, Zhang M, Zhang N | title = Modified Northern blot protocol for easy detection of mRNAs in total RNA using radiolabeled probes | journal = BMC Genomics | volume = 23 | issue = 1 | pages = 66 | date = January 2022 | pmid = 35057752 | pmc = 8772191 | doi = 10.1186/s12864-021-08275-w | doi-access = free }}</ref><ref>{{cite journal | vauthors = Wesierska-Gadek J, Wang ZQ, Schmid G | title = Reduced stability of regularly spliced but not alternatively spliced p53 protein in PARP-deficient mouse fibroblasts | journal = Cancer Research | volume = 59 | issue = 1 | pages = 28–34 | date = January 1999 | pmid = 9892179 | url = https://aacrjournals.org/cancerres/article/59/1/28/505067/Reduced-Stability-of-Regularly-Spliced-but-not }}</ref>

Another approach for measuring mRNA abundance is RT-qPCR. In this technique, [[reverse transcription]] is followed by [[quantitative PCR]]. Reverse transcription first generates a DNA template from the mRNA; this single-stranded template is called [[Complementary DNA|cDNA]]. The cDNA template is then amplified in the quantitative step, during which the [[fluorescence]] emitted by labeled [[hybridization probe]]s or [[Intercalation (biochemistry)|intercalating dyes]] changes as the [[Polymerase chain reaction|DNA amplification]] process progresses.<ref>{{cite journal | vauthors = Padhi BK, Singh M, Rosales M, Pelletier G, Cakmak S | title = A PCR-based quantitative assay for the evaluation of mRNA integrity in rat samples | journal = Biomolecular Detection and Quantification | volume = 15 | pages = 18–23 | date = May 2018 | pmid = 29922590 | pmc = 6006387 | doi = 10.1016/j.bdq.2018.02.001 }}</ref> With a carefully constructed standard curve, qPCR can produce an absolute measurement of the number of copies of original mRNA, typically in units of copies per nanolitre of homogenized tissue or copies per cell.<ref>{{Cite book | vauthors = Kwon YM, Ricke SC   |url=https://link.springer.com/book/10.1007/978-1-61779-089-8 |title=High-Throughput Next Generation Sequencing  |date=2011-03-22 |publisher=[[Springer Science+Business Media]] |isbn=978-1-61779-089-8 |edition=1st |series=Methods in Molecular Biology |volume=733 |language=en |doi=10.1007/978-1-61779-089-8 |eissn=1940-6029 |issn=1064-3745}}</ref> qPCR is very sensitive (detection of a single mRNA molecule is theoretically possible), but can be expensive depending on the type of reporter used; fluorescently labeled oligonucleotide probes are more expensive than non-specific intercalating fluorescent dyes.<ref>{{cite journal | vauthors = Rahmasari R, Raekiansyah M, Azallea SN, Nethania M, Bilqisthy N, Rozaliyani A, Bowolaksono A, Sauriasari R | title = Low-cost SYBR Green-based RT-qPCR assay for detecting SARS-CoV-2 in an Indonesian setting using WHO-recommended primers | journal = Heliyon | volume = 8 | issue = 11 | pages = e11130 | date = November 2022 | pmid = 36339747 | pmc = 9617658 | doi = 10.1016/j.heliyon.2022.e11130 | doi-access = free | bibcode = 2022Heliy...811130R }}</ref>

For [[expression profiling]], or high-throughput analysis of many genes within a sample, [[quantitative PCR]] may be performed for hundreds of genes simultaneously in the case of low-density arrays.<ref>{{cite journal | vauthors = Liang M, Cowley AW, Greene AS | title = High throughput gene expression profiling: a molecular approach to integrative physiology | journal = The Journal of Physiology | volume = 554 | issue = Pt 1 | pages = 22–30 | date = January 2004 | pmid = 14678487 | pmc = 1664740 | doi = 10.1113/jphysiol.2003.049395 }}</ref> A second approach is the [[DNA microarrays|hybridization microarray]]. A single array or "chip" may contain probes to determine transcript levels for every known gene in the genome of one or more organisms.<ref>{{cite journal | vauthors = Bumgarner R | title = Overview of DNA microarrays: types, applications, and their future | journal = Current Protocols in Molecular Biology | volume = Chapter 22 | pages = Unit 22.1 | date = January 2013 | pmid = 23288464 | pmc = 4011503 | doi = 10.1002/0471142727.mb2201s101 }}</ref> Alternatively, "tag based" technologies like [[Serial analysis of gene expression]] (SAGE) and [[RNA-Seq]], which can provide a relative measure of the cellular [[concentration]] of different mRNAs, can be used.<ref>{{cite journal | vauthors = Byron SA, Van Keuren-Jensen KR, Engelthaler DM, Carpten JD, Craig DW | title = Translating RNA sequencing into clinical diagnostics: opportunities and challenges | journal = Nature Reviews. Genetics | volume = 17 | issue = 5 | pages = 257–271 | date = May 2016 | pmid = 26996076 | pmc = 7097555 | doi = 10.1038/nrg.2016.10 }}</ref> An advantage of tag-based methods is the "open architecture", allowing for the exact measurement of any transcript, with a known or unknown sequence.<ref>{{cite journal | vauthors = Meyers BC, Galbraith DW, Nelson T, Agrawal V | title = Methods for transcriptional profiling in plants. Be fruitful and replicate | journal = Plant Physiology | volume = 135 | issue = 2 | pages = 637–652 | date = June 2004 | pmid = 15173570 | pmc = 514100 | doi = 10.1104/pp.104.040840 }}</ref> Next-generation sequencing (NGS) such as [[RNA-Seq]] is another approach, producing vast quantities of sequence data that can be matched to a reference genome. Although NGS is comparatively time-consuming, expensive, and resource-intensive, it can identify [[single-nucleotide polymorphisms]], splice-variants, and novel genes, and can also be used to profile expression in organisms for which little or no sequence information is available.<ref>{{cite journal | vauthors = Kukurba KR, Montgomery SB | title = RNA Sequencing and Analysis | journal = Cold Spring Harbor Protocols | volume = 2015 | issue = 11 | pages = 951–969 | date = April 2015 | pmid = 25870306 | pmc = 4863231 | doi = 10.1101/pdb.top084970 }}</ref>