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

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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Selection and validation of reference genes for quantitative gene expression studies by real-time PCR in eggplant (Solanum melongena L)

BMC Research Notes ◽

10.1186/1756-0500-6-312 ◽

2013 ◽

Vol 6 (1) ◽

pp. 312 ◽

Cited By ~ 47

Author(s):

Nagavara Gantasala ◽

Pradeep Papolu ◽

Prasoon Thakur ◽

Divya Kamaraju ◽

Rohini Sreevathsa ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Real Time Pcr ◽

Reference Genes ◽

Solanum Melongena ◽

Quantitative Gene Expression ◽

Expression Studies ◽

Gene Expression Studies

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Validation of Reference Genes for Relative Quantitative Gene Expression Studies in Cassava (Manihot esculenta Crantz) by Using Quantitative Real-Time PCR

Frontiers in Plant Science ◽

10.3389/fpls.2016.00680 ◽

2016 ◽

Vol 7 ◽

Cited By ~ 8

Author(s):

Meizhen Hu ◽

Wenbin Hu ◽

Zhiqiang Xia ◽

Xincheng Zhou ◽

Wenquan Wang

Keyword(s):

Gene Expression ◽

Real Time ◽

Real Time Pcr ◽

Reference Genes ◽

Manihot Esculenta ◽

Quantitative Real Time Pcr ◽

Manihot Esculenta Crantz ◽

Quantitative Gene Expression ◽

Expression Studies ◽

Gene Expression Studies

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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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Validation of Reference Genes for Quantitative Gene Expression Studies in Octopus minor

Ocean Science Journal ◽

10.1007/s12601-020-0007-9 ◽

2020 ◽

Vol 55 (1) ◽

pp. 183-191

Author(s):

Ilson Whang ◽

Hyun-Sil Kang ◽

Yucheol Kim

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Quantitative Gene Expression ◽

Expression Studies ◽

Gene Expression Studies ◽

Octopus Minor

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Gene Regulation during the Erythrocytic Differentiation of Embryonic Stem Cells.

Blood ◽

10.1182/blood.v106.11.4255.4255 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4255-4255

Author(s):

Ewa Carrier ◽

Shermila Kausal ◽

Anand S. Srivastava

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Growth Factors ◽

Es Cells ◽

Embryonic Stem ◽

Globin Genes ◽

Rt Pcr ◽

Expression Studies ◽

Gene Expression Studies

Abstract We have studied the in vitro differentiation of murine embryonic stem cells (ES cells) towards erythropoiesis and expression of genes during this process. It has been reported that dexamethasone directs ES cells towards erythrocytic differentiation but the mechanism of gene regulation induced by dexamethasone is not well understood. We hypothesized that dexamethasone induces upregulation of erythropoietic genes such as GATA-1, FLK-1, EPO-R and directs ES cells towards erythropoietic differentiation. Murine ES cells (129 CCE) obtained from Dr. Nagy laboratory, Canada (Nagy et al., Histochem Cell Biol., 2001; 115:49–58) were subjected to the in vitro primary hematopoietic differentiation media containing methylcellulose, IMDM, IL -3, IL-6 and SCF (stem cell factor) without LIF (leukemia inhibitory factor) to promote embryoid body (EB) formation. Total RNA was collected on day 3, 5 and 9 EBs for gene expression studies using RT-PCR. On day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combination 1) SCF, EPO, dexamethasone, IGF, 2) SCF, IL-3, IL-6, TPO, 3) SCF IL-3, IL-6, TPO, EPO. Total RNA from day12 of secondary differentiated ES cells was collected to study cytokines and growth factors dependent erythrocytic differentiation and gene regulation, using RT-PCR. Our results demonstrate upregulation of Gata-1, Flk-1, HoxB-4, Epo-R and globin genes (α-globin, BH-1 globin, β-major globin, e -globin and z-globin) in the 9 days old EBs, whereas, RNA collected from 5 days old EBs showed expression of HoxB-4, e-globin, γ-globin, BH1-globin and FLK-1. Three days old EBs showed only HoxB-4 and FLK-1 gene expression and lack of expression of globin genes, indicating that erythtropoiesis-specific genes activate later. Gene expression studies of RNA collected from secondary differentiated ES cells and media containing dexamethasone showed downregulation of GATA-3 and upregulation of GATA-1, Flk-1 and Epo-R in comparison to the two other cytokines and growth factors media combination. These results confirm our hypothesis that dexamethasome induces erythropoiesis by down regulating GATA -3 and upregulating erythropoietic-related genes such as GATA-1, Flk-1 and Epo-R. The morphological characteristics of cells after secondary differentiation showed enhanced production of erythrocytic precursors in dexamethasone containing media, which corresponded with molecular studies. Further studies will address the role of wnt/β-catenin and E-cadherin in this process.

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