RNA extraction for RNA sequencing of archival renal tissues

Landolt, Lea; Marti, Hanspeter; Beisland, Christian; Eikrem, Øystein

doi:10.1080/00365513.2016.1177660

Cited by 45 publications

(43 citation statements)

References 84 publications

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“…The RINe has been widely used as an indicator of RNA quality in NGS, microarray, and qPCR [5][6][7]. However, the DV200 is more suitable than the RINe for quantification of RNA because it can be applied to evaluate not only RNAs extracted from fresh or frozen samples but also samples with lower RINe values, such as RNAs extracted from FFPE samples [8,9]. In our study, the DV200 showed better correlation with the amount of the 1 st NGS library product compared with the RINe even for low-quality samples such as FFPE.…”

Section: Discussionmentioning

confidence: 99%

DV200 Index for Assessing RNA Integrity in Next-Generation Sequencing

Matsubara

Soh

Morita

et al. 2020

BioMed Research International

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Poor quality of biological samples will result in an inaccurate analysis of next-generation sequencing (NGS). Therefore, methods to accurately evaluate sample integrity are needed. Among methods for evaluating RNA quality, the RNA integrity number equivalent (RINe) is widely used, whereas the DV200, which evaluates the percentage of fragments of >200 nucleotides, is also used as a quality assessment standard. In this study, we compared the RINe and DV200 RNA quality indexes to determine the most suitable RNA index for the NGS analysis. Seventy-one RNA samples were extracted from formalin-fixed paraffin-embedded tissue samples (n = 30), fresh-frozen samples (n = 25), or cell lines (n = 16). After assessing RNA quality using the RINe and DV200, we prepared two kinds of stranded mRNA sequencing libraries. Finally, we calculated the correlation between each RNA quality index and the amount of library product (1 st PCR product per input RNA). The DV200 measure showed stronger correlation with the amount of library product than the RINe (R 2 = 0:8208 for the DV200 versus 0.6927 for the RINe). Receiver operating characteristic curve analyses revealed that the DV200 was the better marker for predicting efficient library production than the RINe using a threshold of >10 ng/ng for the amount of the 1 st PCR product per input RNA (cutoff value for the RINe and DV200, 2.3 and 66.1%; area under the curve, 0.99 and 0.91; sensitivity, 82% and 92%; and specificity, 93% and 100%, respectively). Our results indicate that NGS libraries prepared using RNA samples with the DV200 value > 66:1% exhibit greater sensitivity and specificity than those prepared with the RINe values > 2:3. These findings suggest that the DV200 is superior to the RINe, especially for low-quality RNA, because it is a more consistent assessment of the amount of the 1 st NGS library product per input.

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Section: Discussionmentioning

confidence: 99%

DV200 Index for Assessing RNA Integrity in Next-Generation Sequencing

Matsubara

Soh

Morita

et al. 2020

BioMed Research International

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“…For the comparison of different studies using human FFPE renal biopsies for glomerular micro-dissection, we performed a literature search in PubMed looking for articles published until March 2017. There are five studies using different reference transcripts or genes (GAPDH, ACTB and Eukaryotic 18S rRNA) [ 9 – 13 ]. Detailed differences between these studies and our study are highlighted in Table 3 .…”

Section: Resultsmentioning

confidence: 99%

“…Despite the certain degradation of our RNA samples, we had a similar range for RNA purity in between different samples as well as between different amounts of RNA, which seems to be valuable for further analyses [ 30 ]. As shown in Table 3 , one recent study performed a comprehensive analysis for RNA purity, quality and quantity [ 9 ]. Beside this study no other documents the A260/A280 ratio, which might be due to the fact that it is difficult or even impossible to obtain reliable absorption values at A260 in micro-dissected FFPE tissue [ 31 ].…”

Section: Discussionmentioning

confidence: 99%

“…A comprehensive study for RNA quality and quantity was recent performed for the first time in renal FFPE tissue [ 9 ]. Furthermore, six studies [ 9 – 13 ] including ours [ 14 ] have performed successful mRNA gene expression analysis from micro-dissected FFPE glomerular transections using different methodological approaches. Another study described a selection of reference transcripts for mRNA quantification from these specimen [ 15 ].…”

Section: Introductionmentioning

confidence: 99%

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Recommendations for mRNA analysis of micro-dissected glomerular tufts from paraffin-embedded human kidney biopsy samples

et al. 2018

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BackgroundGlomeruli are excellent pre-determined natural structures for laser micro-dissection. Compartment-specific glomerular gene expression analysis of formalin-fixed paraffin-embedded renal biopsies could improve research applications. The major challenge for such studies is to obtain good-quality RNA from small amounts of starting material, as applicable for the analysis of glomerular compartments. In this work, we provide data and recommendations for an optimized workflow of glomerular mRNA analysis.ResultsWith a proper resolution of the camera and screen provided by the next generation of micro-dissection systems, we are able to separate parietal epithelial cells from glomerular tufts. Selected compartment-specific transcripts (WT1 and GLEPP1 for glomerular tuft as well as PAX2 for parietal epithelial cells) seem to be reliable discriminators for these micro-dissected glomerular substructures. Using the phenol–chloroform extraction and hemalaun-stained sections (2 µm), high amounts of Bowman’s capsule transections (> 300) reveal sufficient RNA concentrations (> 300 ng mRNA) for further analysis. For comparison, in unstained sections from a number of 60 glomerular transections upwards, a minimum amount of 157 ng mRNA with a reasonable mRNA purity [A260/A280 ratio of 1.5 (1.4/1.7) median (25th/75th percentiles)] was reversely transcribed into cDNA. Comparing the effect of input RNA (20, 60, 150 and 300 micro-dissected glomerular transections), transcript expression of POLR2A significantly correlated when 60 and 150 laser micro-dissected glomerular transections were used for analysis. There was a lower inter-assay coefficient of variability for ADAMTS13, when at least 60 glomerular transections were used. According to the algorithms of geNormPlus and NormFinder, PGK1 and PPIA are more stable glomerular reference transcripts compared to GUSB, GAPDH, POLR2A, RPLPO, TBP, B2M, ACTB, 18SrRNA and HMBS.ConclusionsOur approach implements compartment-specific glomerular mRNA expression analysis into research applications, even regarding glomerular substructures like parietal epithelial cells. We recommend using of at least 60 micro-dissected unstained glomerular or 300 hemalaun-stained Bowman’s capsule transections to obtain sufficient input mRNA for reproducible results. Hereby, the range of RNA concentrations in 60 micro-dissected glomeruli is low and appropriate normalization of Cq values using our suggested reference transcripts (PGK1 and PPIA) allows compensation with respect to different amounts of RNA purity and quantity.Electronic supplementary materialThe online version of this article (10.1186/s12867-018-0103-x) contains supplementary material, which is available to authorized users.

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“…Since there is no single exclusive marker for CESC identification, confirmation of enrichment after LCM was possible only by RT‐PCR. Methods are available to isolate good quality RNA from laser capture microdissected cells from fresh cryosections (Bath et al, ) or from formalin fixed tissue cross sections (Landolt, Marti, Beisland, Flatberg, & Eikrem, ) and hence LCM of Giemsa stained cytospin smears of limbal basal epithelial cells was adopted in this study. The RNA extracted from such cells captured by LCM had RIN values in the range 4–7.4 (data not shown).…”

Section: Discussionmentioning

confidence: 99%

A novel method for a high enrichment of human corneal epithelial stem cells for genomic analysis

Kasinathan

Namperumalsamy

Muthukkaruppan

et al. 2016

Microscopy Res & Technique

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Understanding the molecular mechanisms that regulate the corneal epithelial stem cells (CESCs) in maintaining corneal homeostasis remains elusive largely due to the lack of a specific marker for their isolation. This study aims to enrich CESCs from human donor limbal epithelium and to evaluate the level of enrichment based on expression of ΔNp63α, a putative CESC marker. A two-stage enrichment of CESCs was carried out. (a) The limbal basal epithelial cells were isolated by differential enzymatic treatment and five-fold enrichment was achieved from 2% of CESCs present in the total limbal epithelium. The CESCs were quantified on the basis of two parameters-high expression of p63/ABCG2 and nucleus to cytoplasmic (N/C) ratio ≥0.7. (b) Cytospin smears of isolated basal cells were Giemsa stained and cells with N/C ratio ≥0.7 were separated by laser capture microdissection. This strategy resulted in an enrichment of CESCs to 78.57% based on two-parameter analysis using p63 and 76.66% using ABCG2. RT-PCR was carried out for ΔNp63 isoforms (α, β, and γ) and connexin-43, with GAPDH for normalization. The expression of ΔNp63α was restricted to the enriched population of CESCs in contrast to its absence in limbal basal cells with N/C ratio <0.7 and CCECs. The unique expression of ΔNp63α and 5.9-fold reduced connexin-43 expression in the enriched population of CESCs indicates its high purity. Further analysis of these cells will help in elucidating the molecular mechanisms associated with stemness and also in identifying a specific marker for CESCs.

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RNA extraction for RNA sequencing of archival renal tissues

Abstract: Total RNA can be extracted from archival renal biopsies in sufficient quality and quantity from one human kidney biopsy section and from around 100 LCM glomerular cross-sections to enable successful RNA library preparation and sequencing using commercially available RNA extraction kits.

Cited by 45 publications

References 84 publications

DV200 Index for Assessing RNA Integrity in Next-Generation Sequencing

DV200 Index for Assessing RNA Integrity in Next-Generation Sequencing

Recommendations for mRNA analysis of micro-dissected glomerular tufts from paraffin-embedded human kidney biopsy samples

A novel method for a high enrichment of human corneal epithelial stem cells for genomic analysis

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