A consideration of the ribonucleic acid depolymerase‐inhibitor systems of mouse tissues

Ellem, K.A.O.; Colter, John S.

doi:10.1002/jcp.1030580308

Cited by 11 publications

(2 citation statements)

References 21 publications

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“…In addition, one needs to be cognizant of changes that can occur in the clinic during surgery, tissue acquisition, and in the pathology laboratory, including; time to freezing, freezing method, tissue type, storage, and the effects of endogenous proteases and RNases. More information on these topics and a general review can be found at the National Cancer Institute Office of Biorepositories and Biospecimen Research (http://biospecimens.cancer.gov/) and is addressed by other researchers22–34.…”

Section: Tissue Considerationsmentioning

confidence: 99%

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Quantitative RT-PCR gene expression analysis of laser microdissected tissue samples

et al. 2009

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Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a valuable tool for measuring gene expression in biological samples. However, unique challenges are encountered when studies are performed on cells microdissected from tissues derived from animal models or the clinic, including specimen related issues, variability of RNA template quality and quantity, and normalization. qRT-PCR using small amounts of mRNA derived from dissected cell populations requires adaptation of standard methods to allow meaningful comparisons across sample sets. The protocol described here presents the rationale, technical steps, normalization strategy, and data analysis necessary to generate reliable gene expression measurements of transcripts from dissected samples. The entire protocol from tissue microdissection through qRT-PCR analysis requires approximately 16 hours.

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Section: Tissue Considerationsmentioning

confidence: 99%

“…In addition, certain organ types may also contain higher level of RNases. Brain tissue inherently contains less RNases than other tissues, with pancreas, spleen, and intestinal mucosa having the most34. Another step in the protocol to examine is the dissection process.…”

Section: ? Troubleshootingmentioning

confidence: 99%

Quantitative RT-PCR gene expression analysis of laser microdissected tissue samples

et al. 2009

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Immuno-Guided Laser-Capture Microdissection of Glial Cells for mRNA Analysis

Nicot¹,

Rambeau²,

Guillot³

et al. 2018

Methods in Molecular Biology

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Laser-capture microdissection (LCM) allows for retrieval of specific cell populations in situ. By combining immunofluorescent labeling with LCM, mRNAs can be probed by qRT-PCR for determining in situ gene expression during health and disease. This approach permits obtaining and analyzing histologically enriched cell populations in a tissue that can be hardly obtained from other methods such as white matter astrocytes from rodents or any individual cell population from archival human or rodent brain tissues. Herein, we present our methodology of laser-captured mouse spinal cord white matter astrocytes, which can be adapted for any cell type in CNS tissue and low RNAse containing tissues. The methods presented with an emphasis on tips and advices include the cryostat section preparation from snap-frozen tissue, an adapted immunofluorescent labeling, a brief overview of LCM using a UV-based technology with polyethylene membrane glass slides, procedures for direct use of RNA from lysis buffer vs. column-based purified RNA, RNA quality/quantity assessment, the reverse transcription and preamplification steps used before real-time qPCR analysis.

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Ribonucleasen

Siebert¹

1966

Enzyme

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A consideration of the ribonucleic acid depolymerase‐inhibitor systems of mouse tissues

Cited by 11 publications

References 21 publications

Quantitative RT-PCR gene expression analysis of laser microdissected tissue samples

Quantitative RT-PCR gene expression analysis of laser microdissected tissue samples

Immuno-Guided Laser-Capture Microdissection of Glial Cells for mRNA Analysis

Ribonucleasen

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