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

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

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab150 ◽

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).

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

Communications Biology ◽

10.1038/s42003-020-01573-1 ◽

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.

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

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2018-0172-oa ◽

2019 ◽

Vol 143 (9) ◽

pp. 1106-1118 ◽

Cited By ~ 10

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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RNA profile diversity across arthropoda: guidelines, methodological artifacts, and expected outcomes

Biology Methods and Protocols ◽

10.1093/biomethods/bpy012 ◽

2018 ◽

Vol 3 (1) ◽

Cited By ~ 4

Author(s):

Danielle M DeLeo ◽

Jorge L Pérez-Moreno ◽

Hernán Vázquez-Miranda ◽

Heather D Bracken-Grissom

Keyword(s):

High Throughput ◽

General Pattern ◽

High Quality ◽

Banding Patterns ◽

Rna Integrity ◽

Rna Quality ◽

Rna Integrity Number ◽

Arthropod Species ◽

Quality Assessments

Abstract High-quality RNA is an important precursor for high-throughput RNA sequencing (RNAseq) and subsequent analyses. However, the primary metric used to assess RNA quality, the RNA Integrity Number (RIN), was developed based on model bacterial and vertebrate organisms. Though the phenomenon is not widely recognized, invertebrate 28S ribosomal RNA (rRNA) is highly prone to a form of denaturation known as gap deletion, in which the subunit collapses into two smaller fragments. In many nonmodel invertebrates, this collapse of the 28S subunit appears as a single band similar in size to the 18S rRNA subunit. This phenomenon is hypothesized to be commonplace among arthropods and is often misinterpreted as a “degraded” rRNA profile. The limited characterization of gap deletion in arthropods, a highly diverse group, as well as other nonmodel invertebrates, often biases RNA quality assessments. To test whether the collapse of 28S is a general pattern or a methodological artifact, we sampled more than half of the major lineages within Arthropoda. We found that the 28S collapse is present in ∼90% of the species sampled. Nevertheless, RNA profiles exhibit considerable diversity with a range of banding patterns. High-throughput RNAseq and subsequent assembly of high-quality transcriptomes from select arthropod species exhibiting collapsed 28S subunits further illustrates the limitations of current RIN proxies in accurately characterizing RNA quality in nonmodel organisms. Furthermore, we show that this form of 28S denaturation, which is often mistaken for true “degradation,” can occur at relatively low temperatures.

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

PeerJ ◽

10.7717/peerj.4664 ◽

2018 ◽

Vol 6 ◽

pp. e4664 ◽

Cited By ~ 8

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.

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PERBANDINGAN TIGA KIT EKSTRAKSI RNA UNTUK ANALISIS TRANSKRIPTOMIKA PADA KELAPA SAWIT (Elaeis guineensis Jacq.)

Jurnal Bioteknologi & Biosains Indonesia (JBBI) ◽

10.29122/jbbi.v6i1.3372 ◽

2019 ◽

Vol 6 (1) ◽

pp. 118

Author(s):

Siti Zulaeha ◽

Devit Purwoko ◽

Imam Cartealy ◽

Teuku Tajuddin ◽

. Karyanti ◽

...

Keyword(s):

Somatic Embryo ◽

Oil Palm ◽

Elaeis Guineensis ◽

Early Stage ◽

Rna Extraction ◽

Transcriptomic Analysis ◽

Total Rna ◽

Rna Integrity ◽

Rna Quality ◽

Rna Integrity Number

Comparison of Three RNA Extraction Kits for Transcriptome Analysis of Oil Palm (Elaeis guineensis Jacq.) ABSTRACTObtaining high-quality RNA is very important at an early stage of molecular biology research. To isolate RNA, high skill and caution are required in following laboratory procedures because RNA is easily degraded, especially samples from plant tissue culture. One of the parameters used to check the total RNA quality is RIN (RNA Integrity Number). The aim of this study was to obtain RNA extraction methods on oil palm leaves, callus and somatic embryos that were of good quality and high concentrations for transcriptomic analysis. RNA extraction was carried out using Plant RNA PureLink (Ambion), Genezol RNA Extraction (Geneaid) and RibospinTM Plant (Geneall) kit methods. The results showed that oil palm leaf, callus and somatic embryo RNA were successfully extracted using the RibospinTM (Geneall) kit. Based on the total RNA number of more than 4 μg and the RIN value of more than 7, the extracted RNA could be used in RNA sequencing for transcriptomic analysis. Keywords: callus, oil palm, RNA analysis, RNA quality, somatic embryo ABSTRAKMenghasilkan RNA berkualitas tinggi sangatlah penting pada tahap awal penelitian biologi molekuler. Untuk mengisolasi RNA diperlukan keterampilan dan kehati-hatian tinggi dalam mengikuti prosedur di laboratorium karena RNA lebih mudah terdegradasi, khususnya sampel hasil kultur jaringan tanaman. Salah satu parameter yang digunakan pada pengecekan kualitas RNA total adalah RIN (RNA Integrity Number). Penelitian bertujuan mendapatkan metode ekstraksi RNA pada daun, kalus dan embrio somatik kelapa sawit yang berkualitas baik dan memiliki konsentrasi tinggi untuk analisa transkriptomika. Ekstraksi RNA dilakukan menggunakan metode kit Plant RNA PureLink (Ambion), Genezol RNA Extraction (Geneaid) dan RibospinTM Plant (Geneall). Hasil menunjukkan bahwa RNA daun, kalus dan embrio somatik kelapa sawit telah berhasil diekstraksi dengan menggunakan kit RibospinTM (Geneall). RNA hasil ekstraksi tersebut dapat digunakan untuk sekuensing RNA dengan tujuan analisis transkriptomika, dilihat dari jumlah total RNA yang lebih dari 4 μg dan nilai RIN lebih dari 7. Kata Kunci: analisis RNA, embrio somatic, kalus, kelapa sawit, kualitas RNA

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Preanalytical robustness of blood collection tubes with RNA stabilizers

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2019-0170 ◽

2019 ◽

Vol 57 (10) ◽

pp. 1522-1529 ◽

Cited By ~ 2

Author(s):

Chiara Stellino ◽

Gaël Hamot ◽

Camille Bellora ◽

Johanna Trouet ◽

Fay Betsou

Keyword(s):

Storage Temperature ◽

Regulation Of Gene Expression ◽

Analysis Data ◽

Blood Collection ◽

Becton Dickinson ◽

Rna Integrity ◽

Qrt Pcr ◽

Rna Integrity Number ◽

Significant Difference ◽

Blood Collection Tubes

Abstract Background Efficient blood stabilization is essential to obtaining reliable and comparable RNA analysis data in preclinical operations. PAXgene (Qiagen, Becton Dickinson) and Tempus (Applied Biosystems, Life Technologies) blood collection tubes with RNA stabilizers both avoid preanalytical degradation of mRNA by endogenous nucleases and modifications in specific mRNA concentrations by unintentional up- or down-regulation of gene expression. Methods Sixteen different preanalytical conditions were tested in PAXgene and Tempus blood samples from seven donors: different mixing after collection, different fill volumes and different 24-h transport temperature conditions after collection. RNA was extracted by column-based methods. The quality of the extracted RNA was assessed by spectrophotometric quantification, A260/A280 purity ratio, RNA Integrity Number (Agilent Bioanalyzer), miRNA quantative real time polymerase chain reaction (qRT-PCR) on two target miRNAs (RNU-24 and miR-16), mRNA quality index by qRT-PCR on the 3′ and 5′ region of the GAPDH gene, and the PBMC preanalytical score, based on the relative expression levels of the IL8 and EDEM3 coding genes. Results When PAXgene RNA and Tempus blood collection tubes were used following the manufacturers’ instructions, there was no statistically or technically significant difference in the output RNA quality attributes. However, the integrity of the RNA extracted from Tempus collection tubes was more sensitive to fill volumes and effective inversion, than to storage temperature, while the integrity of RNA extracted from PAXgene collection tubes was more sensitive to effective inversion and storage temperature than to fill volumes. Conclusions Blood collection tubes with different RNA stabilizers present different robustness to common preanalytical variations.

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Stress–response relationships related to ageing and death of orthodox seeds: a study comparing viability and RNA integrity in soya bean (Glycine max) cv. Williams 82

Seed Science Research ◽

10.1017/s0960258520000197 ◽

2020 ◽

Vol 30 (2) ◽

pp. 161-172

Author(s):

Christina Walters ◽

Margaret B. Fleming ◽

Lisa M. Hill ◽

Emma J. Dorr ◽

Christopher M. Richards

Keyword(s):

Degradation Kinetics ◽

Rna Degradation ◽

Soya Bean ◽

Rna Integrity ◽

Viability Loss ◽

Rna Integrity Number ◽

Oxidative Stresses ◽

Degradation Rates ◽

Highly Correlated ◽

Kinetics Of

AbstractCharacterizing non-lethal damage within dry seeds may allow us to detect early signs of ageing and accurately predict longevity. We compared RNA degradation and viability loss in seeds exposed to stressful conditions to quantify relationships between degradation rates and stress intensity or duration. We subjected recently harvested (‘fresh’) ‘Williams 82’ soya bean seeds to moisture, temperature and oxidative stresses, and measured time to 50% viability (P50) and rate of RNA degradation, the former using standard germination assays and the latter using RNA Integrity Number (RIN). RIN values from fresh seeds were also compared with those from accessions of the same cultivar harvested in the 1980s and 1990s and stored in the refrigerator (5°C), freezer (−18°C) or in vapour above liquid nitrogen (−176°C). Rates of viability loss (P50−1) and RNA degradation (RIN⋅d−1) were highly correlated in soya bean seeds that were exposed to a broad range of temperatures [holding relative humidity (RH) constant at about 30%]. However, the correlation weakened when fresh seeds were maintained at high RH (holding temperature constant at 35°C) or exposed to oxidizing agents. Both P50−1 and RIN⋅d−1 parameters exhibited breaks in Arrhenius behaviour near 50°C, suggesting that constrained molecular mobility regulates degradation kinetics of dry systems. We conclude that the kinetics of ageing reactions at RH near 30% can be simulated by temperatures up to 50°C and that RNA degradation can indicate ageing prior to and independent of seed death.

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