Validation of Lab-on-Chip Capillary Electrophoresis Systems for Total RNA Quality and Quantity Control

Fleige, S.; Riedmaier, Irmgard; Pfaffl, Michael W.

doi:10.1080/13102818.2008.10817562

Cited by 14 publications

(9 citation statements)

References 5 publications

(5 reference statements)

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“…RnA concentrations that were determined in the degradation experiments showed that with lower RnA quality the calculated RnA concentration dropped down. these results show, as described earlier (8), that RNA quantification by the experion and the Bioanalyzer is less accurate than by using UV measurement.…”

Section: Determination Of Rna Quantitymentioning

confidence: 56%

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Comparison of Two Available Platforms for Determination of RNA Quality

Riedmaier¹,

Bergmaier²,

Pfaffl³

2010

Biotechnology & Biotechnological Equipment

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Section: Determination Of Rna Quantitymentioning

confidence: 56%

“…the integrity of RnA is a very critical aspect regarding downstream RnA based analysis like microarray technology and real time Rt-qPcR (1,3,5,6,8). low-quality RnA can compromise the results of such experiments.…”

Section: Introductionmentioning

confidence: 99%

Comparison of Two Available Platforms for Determination of RNA Quality

Riedmaier¹,

Bergmaier²,

Pfaffl³

2010

Biotechnology & Biotechnological Equipment

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“…An alternative platform (Experion) is reported to show superior reproducibility and quantity assessment. 9,39,40 The storage and release of quality samples are the sine qua non of a modern biobank, and biobank personnel assume this responsibility on behalf of patient donors, funders, and researchers, including those who ultimately develop new patient interventions. Biospecimen science focuses on improving the quality of a biobank's unique human sample resource, and will henceforth ensure that fewer research studies are discredited because of substandard samples.…”

Section: Discussionmentioning

confidence: 99%

Maintaining Breast Cancer Specimen Integrity and Individual or Simultaneous Extraction of Quality DNA, RNA, and Proteins from Allprotect-Stabilized and Nonstabilized Tissue Samples

Mee

Carroll

Donatello

et al. 2011

Biopreservation and Biobanking

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The Saint James's Hospital Biobank was established in 2008, to develop a high-quality breast tissue BioResource, as a part of the breast cancer clinical care pathway. The aims of this work were: (1) to ascertain the quality of RNA, DNA, and protein in biobanked carcinomas and normal breast tissues, (2) to assess the efficacy of AllPrep(®) (Qiagen) in isolating RNA, DNA, and protein simultaneously, (3) to compare AllPrep with RNEasy(®) and QIAamp(®) (both Qiagen), and (4) to examine the effectiveness of Allprotect(®) (Qiagen), a new tissue stabilization medium in preserving DNA, RNA, and proteins. One hundred eleven frozen samples of carcinoma and normal breast tissue were analyzed. Tumor and normal tissue morphology were confirmed by frozen sections. Tissue type, tissue treatment (Allprotect vs. no Allprotect), extraction kit, and nucleic acid quantification were analyzed by utilizing a 4 factorial design (SPSS PASW 18 Statistics Software(®)). QIAamp (DNA isolation), AllPrep (DNA, RNA, and Protein isolation), and RNeasy (RNA isolation) kits were assessed and compared. Mean DNA yield and A(260/280) values using QIAamp were 33.2 ng/μL and 1.86, respectively, and using AllPrep were 23.2 ng/μL and 1.94. Mean RNA yield and RNA Integrity Number (RIN) values with RNeasy were 73.4 ng/μL and 8.16, respectively, and with AllPrep were 74.8 ng/μL and 7.92. Allprotect-treated tissues produced higher RIN values of borderline significance (P=0.055). No discernible loss of RNA stability was detected after 6 h incubation of stabilized or nonstabilized tissues at room temperature or 4°C or in 9 freeze-thaw cycles. Allprotect requires further detailed evaluation, but we consider AllPrep to be an excellent option for the simultaneous extraction of RNA, DNA, and protein from tumor and normal breast tissues. The essential presampling procedures that maintain the diagnostic integrity of pathology specimens do not appear to compromise the quality of molecular isolates.

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“…This technology recently gained interest in the plant RNA community (reviewed in [33]), but is partly based on the ribosomal peak ratio (28S/18S). Since the relationship between this ratio and mRNA integrity appears to be unclear [40,48,50,51], RIN (RNA Integrity Number [36]) and RQI (RNA Quality Indicator [52]) values that take into account the complete electropherogram were introduced as a more solid measure for RNA integrity. However, these values were initially assigned by using electropherograms of various mammalian tissues to train the software in an adaptive learning approach.…”

Section: Discussionmentioning

confidence: 99%

How to perform RT-qPCR accurately in plant species? A case study on flower colour gene expression in an azalea (Rhododendron simsii hybrids) mapping population

Keyser¹,

Desmet²,

Bockstaele

et al. 2013

BMC Mol Biol

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BackgroundFlower colour variation is one of the most crucial selection criteria in the breeding of a flowering pot plant, as is also the case for azalea (Rhododendron simsii hybrids). Flavonoid biosynthesis was studied intensively in several species. In azalea, flower colour can be described by means of a 3-gene model. However, this model does not clarify pink-coloration. The last decade gene expression studies have been implemented widely for studying flower colour. However, the methods used were often only semi-quantitative or quantification was not done according to the MIQE-guidelines. We aimed to develop an accurate protocol for RT-qPCR and to validate the protocol to study flower colour in an azalea mapping population.ResultsAn accurate RT-qPCR protocol had to be established. RNA quality was evaluated in a combined approach by means of different techniques e.g. SPUD-assay and Experion-analysis. We demonstrated the importance of testing noRT-samples for all genes under study to detect contaminating DNA. In spite of the limited sequence information available, we prepared a set of 11 reference genes which was validated in flower petals; a combination of three reference genes was most optimal. Finally we also used plasmids for the construction of standard curves. This allowed us to calculate gene-specific PCR efficiencies for every gene to assure an accurate quantification. The validity of the protocol was demonstrated by means of the study of six genes of the flavonoid biosynthesis pathway. No correlations were found between flower colour and the individual expression profiles. However, the combination of early pathway genes (CHS, F3H, F3'H and FLS) is clearly related to co-pigmentation with flavonols. The late pathway genes DFR and ANS are to a minor extent involved in differentiating between coloured and white flowers. Concerning pink coloration, we could demonstrate that the lower intensity in this type of flowers is correlated to the expression of F3'H.ConclusionsCurrently in plant research, validated and qualitative RT-qPCR protocols are still rare. The protocol in this study can be implemented on all plant species to assure accurate quantification of gene expression. We have been able to correlate flower colour to the combined regulation of structural genes, both in the early and late branch of the pathway. This allowed us to differentiate between flower colours in a broader genetic background as was done so far in flower colour studies. These data will now be used for eQTL mapping to comprehend even more the regulation of this pathway.

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Validation of Lab-on-Chip Capillary Electrophoresis Systems for Total RNA Quality and Quantity Control

Cited by 14 publications

References 5 publications

Comparison of Two Available Platforms for Determination of RNA Quality

Comparison of Two Available Platforms for Determination of RNA Quality

Maintaining Breast Cancer Specimen Integrity and Individual or Simultaneous Extraction of Quality DNA, RNA, and Proteins from Allprotect-Stabilized and Nonstabilized Tissue Samples

How to perform RT-qPCR accurately in plant species? A case study on flower colour gene expression in an azalea (Rhododendron simsii hybrids) mapping population

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