scholarly journals The spatial RNA integrity number assay for in situ evaluation of transcriptome quality

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Linda Kvastad ◽  
Konstantin Carlberg ◽  
Ludvig Larsson ◽  
Eva Gracia Villacampa ◽  
Alexander Stuckey ◽  
...  
Keyword(s):  
Gene Expression ◽  
Spatial Variation ◽  
Tissue Quality ◽  
Rna Integrity ◽  
Quality Metric ◽  
Rna Quality ◽  
Rna Integrity Number ◽  
Cellular Resolution

AbstractThe RNA integrity number (RIN) is a frequently used quality metric to assess the completeness of rRNA, as a proxy for the corresponding mRNA in a tissue. Current methods operate at bulk resolution and provide a single average estimate for the whole sample. Spatial transcriptomics technologies have emerged and shown their value by placing gene expression into a tissue context, resulting in transcriptional information from all tissue regions. Thus, the ability to estimate RNA quality in situ has become of utmost importance to overcome the limitation with a bulk rRNA measurement. Here we show a new tool, the spatial RNA integrity number (sRIN) assay, to assess the rRNA completeness in a tissue wide manner at cellular resolution. We demonstrate the use of sRIN to identify spatial variation in tissue quality prior to more comprehensive spatial transcriptomics workflows.

scholarly journals A robust method for RNA extraction and purification from a single adult mouse tendon

2018 ◽  
Author(s):  
M Grinstein ◽  
HL Dingwall ◽  
RR Shah ◽  
TD Capellini ◽  
JL Galloway
Keyword(s):  
Gene Expression ◽  
Adult Mouse ◽  
Rna Extraction ◽  
Cost Effective ◽  
Biological Variation ◽  
Mouse Genetics ◽  
Rna Integrity ◽  
Rna Quality ◽  
Rna Analysis ◽  
Rna Integrity Number

AbstractBackgroundMechanistic understanding of tendon molecular and cellular biology is crucial towards furthering our abilities to design new therapies for tendon and ligament injuries and disease. Recent transcriptomic and epigenomic studies in the field have harnessed the power of mouse genetics to reveal new insights into tendon biology. However, many mouse studies pool tendon tissues or use amplification methods to perform RNA analysis, which can significantly increase the experimental costs and limit the ability to detect changes in expression of low copy transcripts.MethodsSingle Achilles tendons were harvested from uninjured, contralateral injured, and wild type mice between 3-5 months of age, and RNA was extracted. RNA Integrity Number (RIN) and concentration were determined, and RT-qPCR gene expression analysis was performed.ResultsAfter testing several RNA extraction approaches on single adult mouse Achilles tendons, we developed a protocol that was successful at obtaining high RIN and sufficient concentrations suitable for RNA analysis. We found that the RNA quality was sensitive to the time between tendon harvest and homogenization, and the RNA quality and concentration was dependent on the duration of homogenization. Using this method, we demonstrate that analysis of Scx gene expression in single mouse tendons reduces the biological variation caused by pooling tendons from multiple mice. We also show successful use of this approach to analyze Sox9 and Col1a2 gene expression changes in injured compared with uninjured control tendons.DiscussionOur work presents a robust, cost-effective, and straightforward method to extract high quality RNA from a single adult mouse Achilles tendon at sufficient amounts for RNA-seq and RT-qPCR. We show this can reduce biological variation and decrease the overall costs associated with experiments. This approach can also be applied to other skeletal tissues as well as precious human samples.

150 Effect of bovine seminal plasma and sperm on endometrial gene expression

2019 ◽  
Vol 31 (1) ◽  
pp. 200
Author(s):  
B. Fernandez-Fuertes ◽  
J. M. Sanchez ◽  
S. Bages ◽  
P. Lonergan
Keyword(s):  
Gene Expression ◽  
Seminal Plasma ◽  
Reproductive Tract ◽  
Epididymal Sperm ◽  
Rna Integrity ◽  
Rna Quality ◽  
Rna Integrity Number ◽  
Maternal Immune System ◽  
Negative Effect

In cattle, most pregnancy losses are sustained before implantation. Many factors are involved in implantation failure, but in mice, pigs and humans there is increased evidence of a role for the maternal immune system and its regulation by seminal plasma (SP). However, there is little evidence for a role of SP in bovine fertility, where dilution or removal of SP before AI is routine. Therefore, the aim of this work was to determine the effect of bull SP or sperm on endometrial gene expression. To this end, 6 heifers were oestrous synchronised and slaughtered 12h after the onset of oestrus. Five endometrial explants from the horn ipsilateral to the preovulatory follicle were obtained from each animal. Explants were incubated with (1) RPMI medium (control); (2) epididymal sperm (106 epididymal sperm mL−1); (3) complete ejaculate (106 ejaculated sperm mL−1+25% SP); (4) ejaculated sperm alone (106 ejaculated sperm mL−1); and (5) SP alone (25% SP). Epididymal sperm were collected and pooled from the cauda epididymis of 3 bulls slaughtered in a commercial abattoir. In addition, complete ejaculates were obtained from 3 other bulls using an artificial vagina. After pooling the samples, a small volume was washed through a density gradient to obtain the ejaculated sperm, and the rest of the ejaculate was filtered to obtain sperm-free SP. The RPMI media was used to dilute sperm and SP to the working concentrations. After 6h of incubation, explants were snap frozen. The RNA quality was assessed with an Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA, USA) before RT-qPCR analysis. Interestingly, SP had a dramatic effect on endometrium RNA integrity, as evidenced by a lower RNA integrity number in explants exposed to a complete ejaculate (2.4±0.14) or SP (2.4±0.06), in comparison with the control, epididymal sperm, or ejaculated sperm treatments (6.7±0.43, 6.9±0.32, 6.7±0.30, respectively; P<0.05). Due to the low RNA quality, those treatments were excluded from further analysis. However, this finding is currently being explored, along with the possibility of this effect being inherent to species that ejaculate intravaginally. We then compared the ability of ejaculated sperm (which have been exposed to SP) and epididymal sperm (which have never had contact with SP) to regulate the endometrial expression of IL1A, IL1B, IL8, IL6, PTGES2, TNFA, and LIF. Although IL6, IL1A and LIF increased in all animals when exposed to either ejaculated or epididymal sperm, there was no effect of treatment. In conclusion, these data did not support the notion that exposure of sperm to SP is important for the immune regulation of the bovine uterus. In addition, the negative effect of SP on the endometrium, together with the fact that bulls are intravaginal ejaculators, suggests that any putative immunoregulatory role of this fluid in the cattle uterus is indirect. Further analysis of the effect of SP on the vagina and cervix will help elucidate whether this response is present in this species and whether it can propagate to more distal regions of the reproductive tract. This work was supported by Science Foundation Ireland (13/IA/1983, 16/IA/4474).

scholarly journals A robust method for RNA extraction and purification from a single adult mouse tendon

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4664 ◽  
Author(s):  
Mor Grinstein ◽  
Heather L. Dingwall ◽  
Rishita R. Shah ◽  
Terence D. Capellini ◽  
Jenna L. Galloway
Keyword(s):  
Gene Expression ◽  
Adult Mouse ◽  
Rna Extraction ◽  
Cost Effective ◽  
Mouse Genetics ◽  
Rna Integrity ◽  
Rna Quality ◽  
Rna Analysis ◽  
Rna Integrity Number ◽  
Extraction And Purification

BackgroundMechanistic understanding of tendon molecular and cellular biology is crucial toward furthering our abilities to design new therapies for tendon and ligament injuries and disease. Recent transcriptomic and epigenomic studies in the field have harnessed the power of mouse genetics to reveal new insights into tendon biology. However, many mouse studies pool tendon tissues or use amplification methods to perform RNA analysis, which can significantly increase the experimental costs and limit the ability to detect changes in expression of low copy transcripts.MethodsSingle Achilles tendons were harvested from uninjured, contralateral injured, and wild type mice between three and five months of age, and RNA was extracted. RNA Integrity Number (RIN) and concentration were determined, and RT-qPCR gene expression analysis was performed.ResultsAfter testing several RNA extraction approaches on single adult mouse Achilles tendons, we developed a protocol that was successful at obtaining high RIN and sufficient concentrations suitable for RNA analysis. We found that the RNA quality was sensitive to the time between tendon harvest and homogenization, and the RNA quality and concentration was dependent on the duration of homogenization. Using this method, we demonstrate that analysis ofScxgene expression in single mouse tendons reduces the biological variation caused by pooling tendons from multiple mice. We also show successful use of this approach to analyzeSox9andCol1a2gene expression changes in injured compared with uninjured control tendons.DiscussionOur work presents a robust, cost-effective, and straightforward method to extract high quality RNA from a single adult mouse Achilles tendon at sufficient amounts for RT-qPCR as well as RNA-seq. We show this can reduce variation and decrease the overall costs associated with experiments. This approach can also be applied to other skeletal tissues, as well as precious human samples.

scholarly journals Biobanking: Objectives, Requirements, and Future Challenges—Experiences from the Munich Vascular Biobank

2019 ◽  
Vol 8 (2) ◽  
pp. 251 ◽  
Author(s):  
Jaroslav Pelisek ◽  
Renate Hegenloh ◽  
Sabine Bauer ◽  
Susanne Metschl ◽  
Jessica Pauli ◽  
...  
Keyword(s):  
Vascular Tissue ◽  
Rna Stability ◽  
Heart Association ◽  
Housekeeping Genes ◽  
Arterial Disease ◽  
Tissue Quality ◽  
Tissue Samples ◽  
Rna Integrity ◽  
Rna Integrity Number ◽  
Over Time

Collecting biological tissue samples in a biobank grants a unique opportunity to validate diagnostic and therapeutic strategies for translational and clinical research. In the present work, we provide our long-standing experience in establishing and maintaining a biobank of vascular tissue samples, including the evaluation of tissue quality, especially in formalin-fixed paraffin-embedded specimens (FFPE). Our Munich Vascular Biobank includes, thus far, vascular biomaterial from patients with high-grade carotid artery stenosis (n = 1567), peripheral arterial disease (n = 703), and abdominal aortic aneurysm (n = 481) from our Department of Vascular and Endovascular Surgery (January 2004–December 2018). Vascular tissue samples are continuously processed and characterized to assess tissue morphology, histological quality, cellular composition, inflammation, calcification, neovascularization, and the content of elastin and collagen fibers. Atherosclerotic plaques are further classified in accordance with the American Heart Association (AHA), and plaque stability is determined. In order to assess the quality of RNA from FFPE tissue samples over time (2009–2018), RNA integrity number (RIN) and the extent of RNA fragmentation were evaluated. Expression analysis was performed with two housekeeping genes—glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin (ACTB)—using TaqMan-based quantitative reverse-transcription polymerase chain reaction (qRT)-PCR. FFPE biospecimens demonstrated unaltered RNA stability over time for up to 10 years. Furthermore, we provide a protocol for processing tissue samples in our Munich Vascular Biobank. In this work, we demonstrate that biobanking is an important tool not only for scientific research but also for clinical usage and personalized medicine.

Lack of correlation between placental gene expression and RNA integrity number (RIN) or time to collection

Placenta ◽  
2014 ◽  
Vol 35 (9) ◽  
pp. A46-A47 ◽  
Author(s):  
Christopher J. Stodgell ◽  
Richard K. Miller ◽  
Linda Salamone ◽  
Jeffrey Murray ◽  
Jia Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Integrity ◽  
Rna Integrity Number

scholarly journals High RNA quality extracted from the tolerant crop Cyamopsis tetragonoloba (L.) despite possession of low RNA integrity number

2021 ◽  
Vol 35 (1) ◽  
pp. 608-618
Author(s):  
Abdel-Rhman Zakaria Gaafar ◽  
Fahad Al-Qurainy ◽  
Aref Alshameri ◽  
Salim Khan ◽  
Mohammad Nadeem ◽  
...  
Keyword(s):  
Cyamopsis Tetragonoloba ◽  
Rna Integrity ◽  
Rna Quality ◽  
Rna Integrity Number

scholarly journals The Biospecimen Preanalytical Variables Program: A Multiassay Comparison of Effects of Delay to Fixation and Fixation Duration on Nucleic Acid Quality

2019 ◽  
Vol 143 (9) ◽  
pp. 1106-1118 ◽  
Author(s):  
Latarsha J. Carithers ◽  
Rachana Agarwal ◽  
Ping Guan ◽  
Hana Odeh ◽  
Michael C. Sachs ◽  
...  
Keyword(s):  
Rna Integrity ◽  
Time Points ◽  
Rna Quality ◽  
Rna Integrity Number ◽  
Dna And Rna ◽  
Specific Effects ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Preanalytical Variability ◽  
Tumor Types

Context.—Despite widespread use of formalin-fixed, paraffin-embedded (FFPE) tissue in clinical and research settings, potential effects of variable tissue processing remain largely unknown.Objective.—To elucidate molecular effects associated with clinically relevant preanalytical variability, the National Cancer Institute initiated the Biospecimen Preanalytical Variables (BPV) program.Design.—The BPV program, a well-controlled series of systematic, blind and randomized studies, investigated whether a delay to fixation (DTF) or time in fixative (TIF) affects the quantity and quality of DNA and RNA isolated from FFPE colon, kidney, and ovarian tumors in comparison to case-matched snap-frozen controls.Results.—DNA and RNA yields were comparable among FFPE biospecimens subjected to different DTF and TIF time points. DNA and RNA quality metrics revealed assay- and time point–specific effects of DTF and TIF. A quantitative reverse transcription–polymerase chain reaction (qRT-PCR) assay was superior when assessing RNA quality, consistently detecting differences between FFPE and snap-frozen biospecimens and among DTF and TIF time points. RNA Integrity Number and DV200 (representing the percentage of RNA fragments longer than 200 nucleotides) displayed more limited sensitivity. Differences in DNA quality (Q-ratio) between FFPE and snap-frozen biospecimens and among DTF and TIF time points were detected with a qPCR-based assay.Conclusions.—DNA and RNA quality may be adversely affected in some tumor types by a 12-hour DTF or a TIF of 72 hours. Results presented here as well as those of additional BPV molecular analyses underway will aid in the identification of acceptable delays and optimal fixation times, and quality assays that are suitable predictors of an FFPE biospecimen's fit-for-purpose.

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