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

2017 ◽  
Vol 42 (4) ◽  
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.

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

Blood ◽  
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.

scholarly journals Evaluation of Different RNA Extraction Methods from Agropatch Suppressor Assay for Small Quantities of Plant Tissue and Their Application for Analysis of Gene Expression

2018 ◽  
Vol 10 (3) ◽  
pp. 348-353
Author(s):  
Amir G. SHAHRIARI ◽  
Aminallah TAHMASEBI
Keyword(s):  
Gene Expression ◽  
Plant Tissue ◽  
Target Genes ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Total Rna ◽  
Expression Studies ◽  
Rna Extraction Methods ◽  
Gene Expression Studies ◽  
Commercial Kit

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.

scholarly journals Big data from small tissues: extraction of high-quality RNA for different plant tissue types during oilseed Brassica spp. seed development for RNA-Sequencing

2019 ◽  
Author(s):  
Laura Siles ◽  
Peter Eastmond ◽  
Smita Kurup
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
Seed Development ◽  
Developmental Stages ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Extraction Methods ◽  
High Quality ◽  
Gene Expression Analyses ◽  
Rna Extraction Methods

AbstractObtaining high-quality RNA for gene expression analyses from different seed tissues is challenging due to the presence of various contaminants, such as polyphenols, polysaccharides and lipids which interfere with RNA extraction methods. At present, the available protocols for extracting RNA from seeds require high amounts of tissue and are mainly focused on extracting RNA from whole seeds. However, extracting RNA at the tissue level enables more detailed studies regarding tissue specific transcriptome during development. Seeds from heart stage embryo to mature developmental stages of Brassica napus and B. oleracea were sampled for isolation of the embryo, endosperm and seed coat tissues. Ovules and gynoecia wall tissue were also collected at the pre-fertilization stage. After testing several RNA extraction methods, E.Z.N.A. Plant RNA Kit and Picopure RNA Isolation kit extraction methods with some modifications, as well as the use of PVPP for seed coats and endosperms at green stages, resulted in high RNA concentrations with clear 28S and 18S bands and high RIN values. Here, we present efficient and reliable RNA extraction methods for different genotypes of Brassica spp for different tissue types during seed development. The high-quality RNA obtained by using these methodologies is suitable for RNA-Sequencing and gene expression analyses.

Evaluation of RNA isolation procedures from human blood and its application for gene expression studies (Sod-1, Sod-2)

2005 ◽  
Vol 347 (1) ◽  
pp. 156-158 ◽  
Author(s):  
Olha Khymenets ◽  
Jordi Ortuño ◽  
Montserrat Fitó ◽  
Ma Isabel Covas ◽  
Magí Farré ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Blood ◽  
Rna Isolation ◽  
Expression Studies ◽  
Gene Expression Studies

259. Reproductive phenotype of the female aromatase overexpressing mouse

2005 ◽  
Vol 17 (9) ◽  
pp. 105
Author(s):  
J. Liew ◽  
A. E. Drummond ◽  
M. E. Jones ◽  
M. Poutanen ◽  
J. K. Findlay
Keyword(s):  
Gene Expression ◽  
Ovarian Function ◽  
Rna Isolation ◽  
Fat Pad ◽  
Vaginal Smears ◽  
Expression Studies ◽  
Abnormal Pattern ◽  
Gene Expression Studies ◽  
Reproductive Phenotype

Aromatase, the product of the Cyp 19 gene, converts androgens to estrogens. The role of estrogens within the ovary has recently been revisited; using the aromatase knockout (ArKO) mouse, we investigated the effect of estrogen deficiency on ovarian function. We now have an aromatase overexpressing (AROM+) female mouse model with elevated levels of estrogens. These mice were fertile and bred with FVB/N wildtype (WT) males, the AROM+ male being infertile. In this study we characterised the reproductive phenotype of the female AROM+ mouse. 5 WT and 10 AROM+ mice, 22–27 weeks of age were used in the study. The mice were subject to vaginal smears and killed during estrus. The ovaries, uterine horns and gonadal fat were collected and weighed. One ovary and the uterine horns were fixed in formalin for histological assessment, while the other ovary was snap frozen in Ultraspec solution for RNA isolation and gene expression studies. Serum was collected for hormone measurements. All AROM+ mice exhibited an abnormal pattern of cycling that in general, alternated between estrus and post-estrus. AROM+ mice were significantly heavier than their WT counterparts (WT 35.28 ± 2.89 g v. AROM+ 43.38 ± 2.11 g, P < 0.05). Ovarian, uterine and gonadal fat pad weights were not significantly different between the 2 groups (ovary: WT 17.4 ± 1.14 mg v. AROM+ 17.9 ± 0.06 mg; uterine horns: WT 89.7 ± 11.40 mg v. AROM+ 92.1 ± 6.64 mg; gonadal fat pads: WT 2.47 ± 0.62 g v. AROM+ 3.46±0.26 g). Histological, gene expression and hormone analyses are in progress. Our preliminary analyses indicated no significant effect of excess estrogen on ovarian, uterine and gonadal fat pad weights, despite the AROM+ mice being heavier. It remains to be determined as to whether the ovaries and uterine horns are histologically normal. Supported by the NHMRC (Regkeys 241000, 338510, 198705)

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

2013 ◽  
Vol 93 (8) ◽  
pp. 961-966 ◽  
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

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

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.

scholarly journals An optimized method to obtain high-quality RNA from different tissues in Lilium davidii var. unicolor

2021 ◽  
Author(s):  
Chunlei Wang ◽  
Xuemei Hou ◽  
Nana Qi ◽  
Changxia Li ◽  
Yanyan Luo ◽  
...  
Keyword(s):  
Low Cost ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Extraction Methods ◽  
High Yield ◽  
Rna Seq ◽  
High Quality ◽  
Ctab Method ◽  
Different Tissues ◽  
Lilium Davidii

Abstract Background: The high quality, high yield and purity RNA samples are essential for subsequent molecular experiments such as RT-PCR, qPCR and RNA-seq. However, harvest high quality RNA samples from different tissues in Lilium davidii var. unicolor is a great challenge due to its polysaccharides, polyphenols and other secondary metabolites. In this study, three RNA extraction methods, namely modified TRIzol method, Kit and CTAB methods were reported to obtain the total RNA from Lilium davidii var. unicolor, and the efficient RNA extraction protocol (modified TRIzol method) was described. Results: A Nano drop spectrophotometer and 1% gel electrophoresis were used to detect the RNA quality and integrity. Compared with Kit and CTAB methods, the higher RNA concentrations from different tissues were obtained and the A260/280 ratios of RNA samples were ranged from1.97 to 2.27 when using the modified TRIzol method. None of intact RNA bands were gained from the modified CTAB method, indicating that the RNA samples isolation were degraded. As a result of the modified TRIzol method, the RNA samples in the different tissues from Lilium presented clear 28S and 18S bands.Conclusions: Here, modified TRIzol method is an easy, efficient, and low-cost method for RNA isolation from Lilium davidii var. unicolor. Thus, modified TRIzol method is sufficient to gain eligible RNA to support further molecular experiments in Lilium davidii var. unicolor.

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