Optimisation of total RNA extraction from bovine oocytes and embryos for gene expression studies and effects of cryoprotectants on total RNA extraction

The agroinfiltration assay provides fast and efficient way to transiently express genes into plant cells by Agrobacterium tumefaciens. Extraction of RNA of high quality and sufficient amounts is prerequisite for gene expression studies such as quantitative Real Time PCR (q-PCR) from infiltrated areas in agropatch suppressor assay with small quantities of plant tissue. To attain prime RNA extraction from small tissues of infiltrated N. benthamiana plants with Potato virus A helper component proteinase viral suppressor protein, the efficiency of three RNA extraction methods (LiCl, TRIzol reagent and commercial kit) was evaluated. The total RNA yield with LiCl method was 2.83 and 33.2-fold greater than that of TRIzol reagent and commercial kit, respectively. Also, total RNA yield using TRIzol reagent was 11.7-fold higher than that with commercial kit. The A260/A280 ratio mean for TRI reagent (1.95) and kit (1.9) extractions were within the optimum range.q-PCR revealed that the cycle threshold values of housekeeping gene, EIF-1α and target genes AGO1 and ATG6 for RNA extracted using LiCl and kit were 1.07 to 1.3 and 1.02 to 1.12 times higher than those evaluated with the TRIzol method. Overall, TRIzol method showed the most effective approach for obtaining RNA from N. benthamiana patches in gene expression studies.

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Optimization of total RNA extraction from Gram-positive bacteria (coagulase-negative staphylococci) for expression studies

Annales UMCS Pharmacia ◽

10.2478/v10080-008-0175-x ◽

2009 ◽

Vol 22 (2) ◽

pp. 35-38

Author(s):

Rafał Sawicki ◽

Magdalena Stankevič ◽

Grażyna Ginalska

Keyword(s):

Rna Extraction ◽

Coagulase Negative Staphylococci ◽

Gram Positive ◽

Total Rna ◽

Gram Positive Bacteria ◽

Expression Studies ◽

Total Rna Extraction

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Evaluation of the quality of RNA extracted from archival FFPE glioblastoma and epilepsy surgical samples for gene expression assays

Journal of Clinical Pathology ◽

10.1136/jclinpath-2017-204969 ◽

2018 ◽

Vol 71 (8) ◽

pp. 695-701 ◽

Cited By ~ 6

Author(s):

Harry R Haynes ◽

Clare L Killick-Cole ◽

Kelly M Hares ◽

Juliana Redondo ◽

Kevin C Kemp ◽

...

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Ffpe Tissue ◽

Tissue Samples ◽

Total Rna ◽

Expression Studies ◽

Significant Difference ◽

Gene Expression Analyses ◽

Gene Expression Studies ◽

And Control

AimsHistopathological tissue samples are being increasingly used as sources of nucleic acids in molecular pathology translational research. This study investigated the suitability of glioblastoma and control central nervous system (CNS) formalin-fixed paraffin embedded (FFPE) tissue-derived RNA for gene expression analyses.MethodsTotal RNA was extracted from control (temporal lobe resection tissue) and glioblastoma FFPE tissue samples. RNA purity (260/280 ratios) was determined and RNA integrity number (RIN) analysis was performed. RNA was subsequently used for RT-qPCR for two reference genes,18SandGAPDH.ResultsReference gene expression was equivalent between control and glioblastoma tissue when using RNA extracted from FFPE tissue, which has key implications for biological normalisation for CNS gene expression studies. There was a significant difference between the mean RIN values of control and glioblastoma FFPE tissue. There was no significant correlation between 260/280 or RIN values versus total RNA yield. The age of the tissue blocks did not influence RNA yield, fragmentation or purity. There was no significant correlation between RIN or 260/280 ratios and mean qPCR cycle threshold for either reference gene.ConclusionsThis study showed that routinely available CNS FFPE tissue is suitable for RNA extraction and downstream gene expression studies, even after 60 months of storage. Substantial RNA fragmentation associated with glioblastoma and control FFPE tissue blocks did not preclude downstream RT-qPCR gene expression analyses. Cross validation with both archival and prospectively collated FFPE specimens is required to further demonstrate that CNS tissue blocks can be used in novel translational molecular biomarker studies.

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Integrated RNA extraction and RT-PCR for semi-quantitative gene expression studies on a microfluidic device

Laboratory Investigation ◽

10.1038/labinvest.2013.76 ◽

2013 ◽

Vol 93 (8) ◽

pp. 961-966 ◽

Cited By ~ 14

Author(s):

Kirsty J Shaw ◽

Elizabeth M Hughes ◽

Charlotte E Dyer ◽

John Greenman ◽

Stephen J Haswell

Keyword(s):

Gene Expression ◽

Microfluidic Device ◽

Rna Extraction ◽

Rt Pcr ◽

Quantitative Gene Expression ◽

Expression Studies ◽

Gene Expression Studies

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High quality RNA from hydroponically grown grapevine roots suitable for gene expression studies

Turkish Journal of Biochemistry ◽

10.1515/tjb-2016-0301 ◽

2017 ◽

Vol 42 (4) ◽

Cited By ~ 1

Author(s):

Synda Chenenaoui ◽

Samia Daldoul ◽

Ahmed Mliki

Keyword(s):

Gene Expression ◽

Rna Extraction ◽

Extraction Procedure ◽

Rna Isolation ◽

Extraction Methods ◽

Purification Step ◽

High Quality ◽

Rna Quality ◽

Expression Studies ◽

Gene Expression Studies

AbstractObjectives:Grapevine root system plays a great role in sensing and adapting to abiotic and biotic stresses. Identification of candidate genes involved in the tolerance to abiotic stress is becoming a crucial strategy to select and breed resilient genotypes. However, obtaining high quality RNA from grapevine roots under hydroponic culture is difficult. Hence, we have developed a new extraction procedure to improve RNA quality for root gene expression studies.Methods:Conventional RNA extraction methods using CTAB are not suitable for gene expression studies and need to be improved. Here we report the application of a CTAB- based method for RNA extraction using an additional clean-up purification step.Results:The RIN value of the resulting RNA indicated that our procedure allowed the purification of high RNA quality and quantity. Hence, the clean-up purification step efficiently eliminated contaminants which inhibit downstream applications. Derived RNA was successfully used for differential gene expression analysis in salt stressed grapevine by Northern Blot hybridizations.Conclusion:In this study, we developed an efficient RNA isolation protocol from hydroponic cultivated grapevine roots which yielded RNA suitable for gene expression studies. This will open large perspectives in grapevine functional genomics with the identification of pertinent genes of agronomic interest.

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How long does the mRNA remains stable in untreated whole bovine blood?

10.21203/rs.3.rs-434702/v2 ◽

2021 ◽

Author(s):

Rodrigo Giglioti ◽

Bianca Tainá Azevedo ◽

Henrique Nunes de Oliveira ◽

Luciana Morita Katiki ◽

Anibal Eugênio Vercesi Filho ◽

...

Keyword(s):

Gene Expression ◽

Messenger Rna ◽

Expression Patterns ◽

Rna Extraction ◽

Bovine Blood ◽

Rna Integrity ◽

Expression Studies ◽

Gene Expression Studies ◽

Altered Gene ◽

The Stability

Abstract Background: High quality and quantity of messenger RNA (mRNA) are required for accuracy of gene expression studies and other RNA-based downstream applications. Since RNA is considered a labile macromolecular prone to degradation, which may result in falsely altered gene expression patterns, several commercial stabilizing reagents have been developed aiming to keep RNA stable for long period. However, for studies involving large number of experimental samples, the high costs related to these specific reagents may constitute a barrier. Methods and Results: In this context the present study was designed aiming to evaluate the stability of mRNA in whole bovine blood collected in EDTA tubes during storage at common fridge (4°C). Whole blood samples were collected from six Holstein calves and submitted to RNA extraction in each different interval: immediately after blood sampling (< 2 h), at 1-day post-sampling (dps), 2 dps, 3 dps, 7 dps and 14dps intervals. RNA integrity and purity were evaluated, and RT-qPCR assays were run using seven different genes (B2M, ACTB, PPIA, GAPDH, YWHAZ, CD4 and IFN-γ) aiming to evaluate the presence of altered gene transcription during storage. All extracted RNA samples presented high purity, while optimal integrity and unaltered gene expression were observed in whole experimental group up to 3 days of storage.Conclusion: Bovine blood RNA remained stable in K3EDTA tubes for 3 days stored at common fridge and can be successfully and accurately used for gene expression studies.

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The Successful Use of Archived Formalin-Fixed Paraffin-Embedded (FFPE) Material for Gene Expression Studies.

Blood ◽

10.1182/blood.v108.11.4637.4637 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4637-4637

Author(s):

Konstantinos Lilakos ◽

Maria K. Angelopoulou ◽

George Georgiou ◽

Vassilios Salpeas ◽

Penelope Korkolopoulou ◽

...

Keyword(s):

Gene Expression ◽

Real Time Pcr ◽

Rna Extraction ◽

Extraction Methods ◽

Ct Number ◽

Expression Studies ◽

Ffpe Tissues ◽

Ffpe Material ◽

Rna Extraction Methods ◽

Gene Expression Studies

Abstract BACKGROUND: FFPE tissues are widely used as a source of genomic DNA (gDNA), as well as for immunohistochemistry. RNA amplification and its usage for gene expression studies from archived FFPE material has been hampered due to RNA degradation. AIM: The description of a protocol for RNA extraction from archived FFPE tissues followed by reverse transcription (RT) and real time PCR and its implementation in the study of reference genes. METHODS: RNA was extracted from single 15μm sections from 18 samples 30 months old and 6 samples 10 years old from patients with diffuse large B-cell lymphoma (DLBCL). The FFPE RNA Extraction kit (Roche) was used, followed by DNA digestion and run on an Agilent2100 Bioanalyzer. RT was performed using the Transcriptor System (Roche) with modified temperatures followed by real time PCR for the c-abl and Survivin genes using the Universal Library probes and primers (Roche). In addition for the 10-year old samples, beta2 microglobulin (b2m) and G6PDH were also amplified. RESULTS: RNA that was yielded, was around 250 bp. c-abl was successfully amplified in all 18 samples up to 30 months old with a median cycle (Ct) number of 33.74. All 3 control genes were successfully amplified from the 10-year old samples with a median Ct number of 30.3 for G6PDH, 23.4 for b2m and 28.6 for c-abl. To further evaluate the amplification potency we successfully amplified Survivin transcripts with a median Ct number of 35.2. Survivin/abl ratio was calculated at a median of 0.5 for all DLBCL samples. gDNA interferenence was excluded, since no amplification signal was observed when gDNA was used as a template. The special RNA extraction methods, elevated temperatures for RT, as well as the use of the Universal Library probes, which are specifically designed to be short (8–9bp) and hybridize to a short amplicon (72bp for c-abl and 97bp for Survivin) were the major novelties of this method. Conclusions: Specialized RNA extraction methods and the use of Universal Library Probes can succesfully lead to several target gene amplification from archived FFPE material and potentially provide templates for gene expression studies.

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