Biobanking of fresh frozen tissue: RNA is stable in nonfixed surgical specimens

Micke, Patrick; Ohshima, Mitsuhiro; Tahmasebpoor, Simin; Ren, Zhonghai; Östman, Arne; Pontén, Fredrik; Botling, Johan

doi:10.1038/labinvest.3700372

Cited by 205 publications

(168 citation statements)

References 22 publications

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“…Furthermore, many published techniques claiming high-quality RNA isolation from FR tissue either relied only on expression of HKGs [2,3] and A 260 /A 280 ratios [34,35] or did not perform RNA quality assessment at all [8,10,36]. Here we show that contrary to previous results with FR tissue [4,[37][38][39], the modified Arcturus RNA extraction method was optimal for the extraction of high-quality RNA compared with the Ambion and Sigma methods for cervical biopsy sections (Fig. 1B).…”

Section: Discussioncontrasting

confidence: 56%

Gene expression analysis of interferon κ in laser capture microdissected cervical epithelium

DeCarlo

Escott

Werner

et al. 2008

Analytical Biochemistry

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Optimal sample handling techniques for tissue preparation and storage, RNA extraction and quantification, and target gene detection are crucial for reliable gene expression analysis. Methods for measuring low-expressing genes, such as interferons, in human cervical samples are not described in the scientific literature. To detect interferon mRNA in human cervical samples we obtained normal and dysplastic frozen and formalin-fixed cervical biopsies from colposcopy. Histopathological diagnosis was performed by one pathologist. Cervical keratinocytes were isolated using laser capture microdissection. Immortalized keratinocytes transduced with or devoid of an HPV oncogene were used for initial method development. RNA from samples was extracted and integrity tested to compare tissue storage and extraction methods. The expression of five housekeeping genes was analyzed in cell lines and patient tissue to permit normalization between samples using quantitative real-time polymerase chain reaction. The usefulness of cDNA amplification was assessed for the detection of low-expressing interferon κ in cervical tissue. Here we report optimal tissue storage conditions, RNA extraction, sample normalization, and transcript amplification, as well as the sensitivity of quantitative real-time polymerase chain reaction and laser capture microdissection, for interferon κ detection in cervical tissue. Without these optimized techniques, interferon κ detection would be unattainable in cervical samples. KeywordsCervical keratinocytes; Frozen and formalin-fixed cervical tissue; Housekeeping genes; Interferons; Interferon κ; Laser capture microdissection; Quantitative real-time polymerase chain reaction Quantitative real-time polymerase chain reaction (qRT-PCR) 1 for the molecular analysis of disease is a powerful and widely used tool. Extraction of high-quality RNA for use in gene expression analysis techniques such as reverse transcription (RT), qRT-PCR, and cDNA microarrays is of great importance. Although obtaining high-quality RNA from cell lines is relatively straightforward, the complexity and heterogeneity of human tissue pose considerable challenges for linking gene expression patterns with disease state. Nonetheless,

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Section: Discussioncontrasting

confidence: 56%

Gene expression analysis of interferon κ in laser capture microdissected cervical epithelium

DeCarlo

Escott

Werner

et al. 2008

Analytical Biochemistry

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“…Unfortunately, these data were inconclusive with a large range of values obtained. While we do not know the reason for this technical difficulty and mRNA is reported to be stable in surgical specimens under the conditions used here, 21 it is possible that the inflammatory responses present in tissues with active disease interfered with mRNA stability or other technical aspects of mRNA detection. We also attempted quantitative real-time RT-PCR using colon biopsies from UC and CD patients.…”

Section: Ileal Epithelial Mlck Expression Is Increased In CDmentioning

confidence: 80%

Epithelial myosin light chain kinase expression and activity are upregulated in inflammatory bowel disease

Blair

Kane

Clayburgh

et al. 2006

Laboratory Investigation

270

198

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The intestinal epithelial barrier is frequently disrupted in inflammatory bowel disease (IBD) and this has been proposed to play a role in disease pathogenesis and reactivation. In vitro studies show that cytokine-induced epithelial barrier dysfunction can be mediated by increased myosin light chain kinase (MLCK) expression and subsequent myosin II regulatory light chain (MLC) phosphorylation. However, this has never been examined in human disease. The aim of these studies, therefore, was to determine whether MLCK is upregulated in the intestinal epithelium of IBD patients. MLCK expression and MLC phosphorylation in human intestinal resection and biopsy specimens were determined by quantitative immunofluorescence microscopy and correlated with clinical and histopathological data. The data show that ileal epithelial MLCK expression was mildly upregulated in inactive IBD. Expression increased further in active disease, with progressive increases in MLCK expression correlating positively with histological disease activity. This correlation between activity and MLCK expression was also seen in individual patients where areas of differing disease activity were analyzed. Colonic epithelial MLCK expression was similarly increased in active IBD and these increases also correlated positively with disease activity, both in individual patients and the overall study group. To evaluate MLCK enzymatic activity, MLC phosphorylation was assessed in snap-frozen colon biopsies. MLC phosphorylation was significantly increased in biopsies with active, but not inactive, IBD. Therefore, these data show that MLCK expression and enzymatic activity are increased in IBD. Moreover, the correlation with disease activity suggests that MLCK upregulation may contribute to barrier dysfunction and IBD pathogenesis.

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“…Although RNA is easily degraded when extracted from small amount of material, intact RNA can be routinely isolated from snap frozen tissues according to previous studies. 5,24 We compared the RNA quality of scrapings to that of the snap frozen tissue samples side by side. All the scraping samples exhibited clear ribosomal RNA peaks and the RIN values above 7, similar to the tissue samples, indicating no major degradation.…”

Section: Discussionmentioning

confidence: 99%

“…Consequently, fresh tissue biobanking from resected breast cancers specimens is a central and given method at most University Hospitals. 3,4 Preserved tissue material may be used for additional clinical investigations on DNA, mRNA, and biomarker protein levels after routine histopathological assessments, [5][6][7] and is a unique source for future research projects. Two different biobanking methods are used for this purpose, although the later is by many institutions considered to be too costly and complicated:…”

mentioning

confidence: 99%

Superficial scrapings from breast tumors is a source for biobanking and research purposes

Fredriksson

Karthik

et al. 2014

Laboratory Investigation

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Breast cancer is a unique tumor disease in terms of the stringent requirement of predictive biomarker assessments. As recommended by current international guidelines, the established markers consist of estrogen receptor (ER), progesterone receptor, human epidermal growth factor and Ki67, and are primarily analyzed by immunohistochemistry. However, new diagnostic methods based on microarray or next-generation sequencing on DNA and mRNA level are gaining ground. These analyses require fresh-frozen tumor tissue that is generally not available from tumors o10 mm in diameter, comprising almost 25% of all resected breast cancer at our department. We here present a simple and standardized method to generate material from small tumors without risking the histopathological examination. Furthermore, we show that the quality of this material is sufficient for subsequent analysis on mRNA, DNA, and epigenetic level. We were also able to use this method for isolation and expansion of cancer stem cells from the majority of tumors. Consequently, researches can be provided with clinically relevant material for translational studies. In conclusion, this method opens up a new possibility for usage of valuable fresh tumor material for research purposes, biobanking, and next-generation sequencing.

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Biobanking of fresh frozen tissue: RNA is stable in nonfixed surgical specimens

Cited by 205 publications

References 22 publications

Gene expression analysis of interferon κ in laser capture microdissected cervical epithelium

Gene expression analysis of interferon κ in laser capture microdissected cervical epithelium

Epithelial myosin light chain kinase expression and activity are upregulated in inflammatory bowel disease

Superficial scrapings from breast tumors is a source for biobanking and research purposes

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