Improvement of extraction and processing of RNA from renal biopsies

Groningen, Marian C. Roos-van; Eikmans, Michael; Baelde, Hans J.; Heer, Emile de; Bruijn, Jan A.

doi:10.1111/j.1523-1755.2004.00366.x

Cited by 30 publications

(23 citation statements)

References 11 publications

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“…42 Of note, SuperScript III was considerably superior to the first generation of SuperScript (data not shown) that we reported on in a previous study. 43 Finally, we showed that RNA degradation has a major negative impact on mRNA expression levels, which were determined by qPCR. This finding is in line with the highly negative correlation found between the RNA integrity number (RNA quality index or RQI in our study) and the Cq value from the reverse transcriptase PCR assay in earlier studies.…”

Section: Discussionmentioning

confidence: 92%

Blood cell mRNAs and microRNAs: optimized protocols for extraction and preservation

Eikmans

Rekers

Anholts

et al. 2013

Blood

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Assessing messenger RNA (mRNA) and microRNA levels in peripheral blood cells may complement conventional parameters in clinical practice. Working with small, precious samples requires optimal RNA yields and minimal RNA degradation. Several procedures for RNA extraction and complementary DNA (cDNA) synthesis were compared for their efficiency. The effect on RNA quality of freeze-thawing peripheral blood cells and storage in preserving reagents was investigated. In terms of RNA yield and convenience, quality quantitative polymerase chain reaction signals per nanogram of total RNA and using NucleoSpin and mirVana columns is preferable. The SuperScript III protocol results in the highest cDNA yields. During conventional procedures of storing peripheral blood cells at -180°C and thawing them thereafter, RNA integrity is maintained. TRIzol preserves RNA in cells stored at -20°C. Detection of mRNA levels significantly decreases in degraded RNA samples, whereas microRNA molecules remain relatively stable. When standardized to reference targets, mRNA transcripts and microRNAs can be reliably quantified in moderately degraded (quality index 4-7) and severely degraded (quality index <4) RNA samples, respectively. We describe a strategy for obtaining high-quality and quantity RNA from fresh and stored cells from blood. The results serve as a guideline for sensitive mRNA and microRNA expression assessment in clinical material.

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Section: Discussionmentioning

confidence: 92%

Blood cell mRNAs and microRNAs: optimized protocols for extraction and preservation

Eikmans

Rekers

Anholts

et al. 2013

Blood

Self Cite

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“…Frozen gastric tissues (100 mg) were normalized in 1 ml TRIzol ® reagent, and total RNA was arranged as previously described (21). The total RNA was measured using a spectrophotometer at 260 nm (VICTOR3; Perkin Elmer, Wellesley, MA, USA), and the RNA was quantified by 1% formaldehyde-agarose gel electrophoresis.…”

Section: Induction Of Gastric Mucosal Lesionsmentioning

confidence: 99%

Protective effect of Acer mono Max. sap on water immersion restraint stress-induced gastric ulceration

Park

Son

et al. 2011

Experimental and Therapeutic Medicine

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Abstract. Acer mono Max. sap (AmMs) is called 'Gol-Li-Su' or 'Go-Lo-Soe' in Korean, which means 'water beneficial to the bones'. It is reported that the sap contains several types of minerals and sugars. In particular, the calcium concentration of the sap is 36.5 times higher than that of commercial mineral water. Apart from its anti-osteoporosis effect, no reports have addressed the biological activities of AmMs against degenerative diseases. In the present study, we investigated whether AmMs alleviates gastric ulcer-related symptoms in a stress-induced mouse model. To assess the effect of AmMs on gastric ulcer-like symptoms, we carried out a water immersion restraint (WIRE) test and found that AmMs has potential in alleviating gastric ulcers in a concentration-dependent manner. These results indicate that the nutritional factors of the sap mitigate the gastric ulcer-related symptoms caused by stress-induced gastric lesions in mice. AmMs-treated mice exhibited a significant decrease in the ulcer index as compared to those treated with omeprazole or L-arginine. To examine one potential mechanism underlying this effect, we performed reverse transcription-polymerase chain reaction to ascertain whether molecular markers were associated with the mitigation of the gastric lesions. Epithelial and/or tissue nitric oxide synthase (NOS) was assessed to determine whether or not the genes were down-regulated dose-dependently by the sap. The levels of these enzymes were found to be lower in the tissue samples treated with AmMs compared with the levels in the control samples. These findings collectively suggest that AmMs significantly protects the gastric mucosa against WIRE stress-induced gastric lesions, at least in part, by alleviating inducible NOS and/or neuronal NOS expression. IntroductionAcute gastric mucosal injury is a serious clinical problem worldwide. The symptoms cause severe gastric ulceritis and, with sustained exposure, eventually lead to gastric cancer. Researchers have developed many in vivo gastric lesion models: the water immersion restraint (WIRE) stress model, the indomethacin-induced model and the ethanol-induced model using animals (1). The WIRE model has previously been used to observe various aspects affecting the formation of and recovery from gastric mucosal lesions, including sulphydryls, prostaglandins, growth factors and polyamines (2). WIRE stress-induced gastric lesions have also been used to study the roles of cell death and gastric acid secretion in ulcerogenesis (3).In general terms, gastric lesions are a disorder of the gastric blockade, which typically protects against cavernous problems caused by hydrogen ions and other toxic substances generated in the lumen (4). One main component of the gastric barrier is gastric microcirculation; a disturbance in gastric mucosal perfusion results in the formation of lesions and ulcers, such as those that present in animal models of ischemic gastric lesions (5). Gastric blood flow is classically controlled by signaling molecules, such as prostagland...

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“…35,36 A third method is the use of preservatives such as RNAlater preservative. [23][24][25][26][27] We demonstrate, for the first time, the concordance in a RHS (the numerical output of a prognostic algorithm) and in the prediction of recurrence when using RNAlater preservative-suspended and snapfrozen tissues (Tables 1 and 2). …”

Section: Rna Integrity and The Sample Handling Problemmentioning

confidence: 99%

“…Ellis and co-workers 25 examined core needle breast biopsies that were snap-frozen or stored in RNAlater preservative. More recently, Alfredson and co-workers 26 isolated RNA from tissues stored in RNAlater preservative, and Roos-van Groningen and co-workers 27 examined the utility of RNAlater preservative in renal cortical tissue. These studies, however, did not systematically compare large numbers of paired tumors (stored in RNAlater and snapfrozen) and most used cDNA arrays of lower densities.…”

mentioning

confidence: 99%

Prognostic Gene Expression Signatures Can Be Measured in Tissues Collected in RNAlater Preservative

Chowdary

Lathrop

Skelton

et al. 2006

The Journal of Molecular Diagnostics

121

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Gene expression signatures have the ability to serve in both prognostic and predictive capacities in patient management. The use of RNA as the starting material and the lability of this analyte, however, dictate that tissues must be snap-frozen or stored in a solution that can maintain the integrity of the RNA. We compared pairs of snap-frozen and RNAlater preservativesuspended tissue from 30 such paired lymph nodenegative breast tumors and 21 such paired Dukes' B colon tumors. We assessed the correlation of gene expression profiles and prediction of recurrence based on two prognostic algorithms. Tissues stored in RNAlater preservative generated expression profiles with excellent correlation (average Pearson correlation coefficients of 0.97 and 0.94 for the breast and colon tumor pairs, respectively) compared to those produced by tissues that were snap-frozen. The correlation in the prediction of recurrence was 97% and 95% for the breast and colon tumor pairs, respectively, between these two types of tissue handling protocols. This novel finding demonstrates that prognostic signatures can be obtained from RNAlater preservative-suspended tissues, an important step in bringing gene expression signatures to the clinic. (J

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Improvement of extraction and processing of RNA from renal biopsies

Cited by 30 publications

References 11 publications

Blood cell mRNAs and microRNAs: optimized protocols for extraction and preservation

Blood cell mRNAs and microRNAs: optimized protocols for extraction and preservation

Protective effect of Acer mono Max. sap on water immersion restraint stress-induced gastric ulceration

Prognostic Gene Expression Signatures Can Be Measured in Tissues Collected in RNAlater Preservative

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