Comparison of Microarray and Quantitative Real-Time PCR Methods for Measuring MicroRNA Levels in MSC Cultures

Camarillo, Cynthia; Swerdel, Mavis R.; Hart, Ronald P.

doi:10.1007/978-1-60761-999-4_30

Cited by 29 publications

(17 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our findings are similar or better than the moderate concordance rates reported by prior studies of comparisons between miR quantitation methods. [33, 34] The direction of differential expression observed in our study using the Firefly Bioworks Circulating microRNA assay was consistent with previous observations for a prior study reporting findings for miR-320a using qPCR-based quantitation. [23]…”

Section: Resultssupporting

confidence: 91%

Circulating microRNA-320a and microRNA-486 predict thiazolidinedione response: Moving towards precision health for diabetes prevention

et al. 2015

View full text Add to dashboard Cite

Introduction The aims of this study were to compare microRNA (miR) expression between individuals with and without insulin resistance and to determine whether miRs predict response to thiazolidinedione treatment. Materials and Methods In a sample of 93 healthy adults, insulin resistance was defined as steady state plasma glucose (SSPG) ≥180mg/dL and insulin sensitive as <120mg/dL. Response to thiazolidinedione therapy was defined as ≥10% decrease in SSPG. We selected a panel of microRNAs based on prior evidence for a role in insulin or glucose metabolism. Fold change and Wilcoxon rank sum tests were calculated for the 25 miRs measured. Results At baseline, 81% (n=75) of participants were insulin resistant. Five miRs were differentially expressed between the insulin resistant and sensitive groups: miR-193b (1.45 fold change (FC)), miR-22-3p (1.15 FC), miR-320a (1.36 FC), miR-375 (0.59 FC), and miR-486 (1.21 FC) (all p<0.05). In the subset who were insulin resistant at baseline and received thiazolidinediones (n=47), 77% (n=36) showed improved insulin sensitivity. Six miRs were differentially expressed between responders compared to non-responders: miR-20b-5p (1.20 FC), miR-21-5p, (0.92 FC), miR-214-3p (1.13 FC), miR-22-3p (1.14 FC), miR-320a (0.98 FC), and miR-486-5p (1.25 FC) (all p<0.05). Discussion This study is the first to report miRs associated with response to a pharmacologic intervention for insulin resistance. MiR-320a and miR-486-5p identified responders to thiazolidinedione therapy among the insulin resistant group.

show abstract

Section: Resultssupporting

confidence: 91%

Circulating microRNA-320a and microRNA-486 predict thiazolidinedione response: Moving towards precision health for diabetes prevention

et al. 2015

View full text Add to dashboard Cite

show abstract

“…We decided to use RT-qPCR, since it is a technique widely used to test gene expression in a more precise, simple and cost effective way compared to other alternative techniques, such as arrays, which show a high variability in signal-to-noise ratio, require properly trained staff and it is more expensive 23 . We hypothesized HPRT1 results obtained by RT-qPCR should be comparable to other housekeeping genes, such as SNRPD3 or Jun.…”

Section: Discussionmentioning

confidence: 99%

Impact of different stabilization methods on RT-qPCR results using human lung tissue samples

Esteva-Socias

Gómez-Romano

Carrillo-Ávila

et al. 2020

Sci Rep

View full text Add to dashboard Cite

Aiming to increase the reproducibility of biomedical research results, biobanks obtain human tissues of the highest quality and carry out different storage methods adapted to the needs of analytical technique to be performed by the biomedical researchers. However, there is much controversy and little data concerning the real impact of different stabilization methods on tissue quality, integrity and functionality of derived biomolecules. The influence of four stabilization methods [RNAlater (RNL), snap freezing (SF), snap freezing using Optimal Cutting Tissue compound (SF-OCT) and formalin-fixed paraffin-embedded (FFPE)] on RNA quality and integrity was evaluated in paired samples of lung tissue. RNA integrity was evaluated through PCR-endpoint assays amplifying six fragments of different length of the HPRT1 gene and RNA Integrity Number (RIN). To evaluate the difference of tissue functionality among the stabilization methods tested, RT-qPCRs were performed focusing on the differential expression of the HPRT1, SNRPD3 and Jun genes. RNA from the samples preserved with the RNL or SF-OCT method showed better integrity compared to SF and FFPE, measured by PCR-endpoint and RT-qPCR assays. However, only statistically significant differences were observed between the RNA from FFPE and other stabilization methods when gene expression of HPRT1, SNRPD3 and Jun housekeeping genes were determined by RT-qPCR. For the three mentioned genes, Cq and RIN values were highly correlated. The present work describes the fragility of SF samples, being critical the moment just before RNA extraction, although further experiments of tissue RNA are needed. Standardization pre-analytic workflow can lead to improved reproducibility between biomedical research studies. The present study demonstrated clear evidences about the impact of the stabilization method on RNA derived from lung human tissue samples.diluted cDNA and 0,6 µl of PCR-grade water. All PCRs were performed in triplicate from one sample taken from each preservation condition in all 20 patients included. PCR reactions were carried out in 96 well plates and using the CFX96 Real-Time PCR Detection System (Bio-Rad) following the steps: 95 °C for 2 minutes, 40 cycles of amplification (denaturation at 95 °C for 5 seconds, annealing for 10 seconds, and extension at 72 °C for 20 seconds) and final melt curve analysis from 65 °C to 95 °C to exclude unspecific PCR products.The reproducibility and dynamic ranges for qPCR assays were determined by constructing a standard curve, beginning with 100 ng of RNA template as initial concentration and then serial diluted 1/5 until five linear concentration points. For Cq values acquisition, RFU threshold value was adjusted to 50 RFUs for each run.Statistics. All data generated from integrity and functional studies of RNA samples were analysed using multiple comparison tests: ANOVA test with Bonferroni correction to detect significant differences between procedures with at least a signification of p value <0.01. The spearman r index between RNA integ...

show abstract

“…In addition, these cells have the capacity for osteogenic, adipogenic and chondrogenic differentiation. However, significant differences in the functional efficacy of MSCs derived from different sources has been suggested by studies showing differences in gene expression and multilineage differentiation dependent on the MSC source of origin [41, 42]. …”

Section: Origin and Identification Of Mscsmentioning

confidence: 99%

Mesenchymal Stem Cells as a Treatment for Peripheral Arterial Disease: Current Status and Potential Impact of Type II Diabetes on Their Therapeutic Efficacy

Yan

Tie

et al. 2013

Stem Cell Rev and Rep

View full text Add to dashboard Cite

Mesenchymal stem cells (MSCs), due to their paracrine, transdifferentiation, and immunosuppressive effects, hold great promise as a therapy for peripheral arterial disease. Diabetes is an important risk factor for peripheral arterial disease; however, little is known of how type II diabetes affects the therapeutic function of MSCs. This review summarizes the current status of preclinical and clinical studies that have been performed to determine the efficacy of MSCs in the treatment of peripheral arterial disease. We also present findings from our laboratory regarding the impact of type II diabetes on the therapeutic efficacy of MSCs neovascularization after the induction of hindlimb ischemia. In our studies, we documented that experimental type II diabetes in db/db mice impaired MSCs’ therapeutic function by favoring their differentiation towards adipocytes, while limiting their differentiation towards endothelial cells. Moreover, type II diabetes impaired the capacity of MSCs to promote neovascularization in the ischemic hindlimb. We further showed that these impairments of MSC function and multipotency were secondary to hyperinsulinemia-induced, Nox4-dependent oxidant stress in db/db MSCs. Should human MSCs display similar oxidant stress-induced impairment of function, these findings might permit greater leverage of the potential of MSC transplantation, particularly in the setting of diabetes or other cardiovascular risk factors, as well as provide a therapeutic approach by reversing the oxidant stress of MSCs prior to transplantation.

show abstract

Comparison of Microarray and Quantitative Real-Time PCR Methods for Measuring MicroRNA Levels in MSC Cultures

Cited by 29 publications

References 29 publications

Circulating microRNA-320a and microRNA-486 predict thiazolidinedione response: Moving towards precision health for diabetes prevention

Circulating microRNA-320a and microRNA-486 predict thiazolidinedione response: Moving towards precision health for diabetes prevention

Impact of different stabilization methods on RT-qPCR results using human lung tissue samples

Mesenchymal Stem Cells as a Treatment for Peripheral Arterial Disease: Current Status and Potential Impact of Type II Diabetes on Their Therapeutic Efficacy

Contact Info

Product

Resources

About