Comparison of two total RNA extraction protocols using the marine gorgonian coral Pseudopterogorgia elisabethae and its symbiont Symbiodinium sp

Santiago-Vázquez, Lory Z.; Ranzer, Llanie K.; Kerr, Russell G.

doi:10.2225/vol9-issue5-fulltext-15

Cited by 26 publications

(19 citation statements)

References 8 publications

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“…The procedures for these 3 methods are summarized in the schematics in Figure 2. Some studies have suggested the yield of RNA obtained using the GITC-based method can be higher than the column based method for some samples [22][23][24][25].…”

Section: Introductionmentioning

confidence: 99%

Extraction of RNA from tough tissues with high proteoglycan content by cryosection, second phase separation and high salt precipitation

2015

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Reverse-transcription quantitative PCR (RT-qPCR) is a powerful technique for quantification of gene expression. Extraction of RNA directly from human and large animal intervertebral disc (IVD) tissue is technically challenging due to its tough nature, low cell-to-matrix ratio and high proteoglycan content. IVD is consisted of nucleus pulposus (NP) and annulus fibrosis (AF). We compared the yield and quality of RNA extracted from cultured cells using TRIzol and combined TRIzol-column method (TRIspin) with and without bovine NP tissue added for high proteoglycan content simulation. Higher yields were obtained using the TRIzol method compared to the TRIspin method and the quality of RNA extracted using both methods was comparable. Cryosectioning was by far the most effective homogenization method for the tough bovine NP tissue. The typical modification to TRIzol extraction by the use of high salt solution in a previously reported study for tissue with high proteoglycan was insufficient for bovine NP tissue, where undesirable precipitates were formed during the RNA precipitation step. This necessitates other modifications to the protocol. In our study, the combination of additional phase separation and high salt precipitation was shown to be effective to avoid the formation of the undesirable precipitates. The modified TRIzol method was compared with the TRIspin method and shown to give higher RNA yield which resulted in smaller Ct values in RT-qPCR. In addition, we showed that the cryosectioning method was applicable to bovine muscle, mouse IVD and rat NP tissues. This method of RNA preparation also allows simultaneous preparation of tissue for histological study and quantitative gene expression analysis.

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Section: Introductionmentioning

confidence: 99%

Extraction of RNA from tough tissues with high proteoglycan content by cryosection, second phase separation and high salt precipitation

2015

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“…from human, animal or plant), the sampling procedures (biopsy material, single cell sampling, laser micro-dissection, tissue) as well as the method of RNA isolation (total RNA or polyadenylated RNA methods) differ very often from one laboratory to another (Bustin et al, 2005;Pfaffl, 2004). As a matter of example the use of liquid extraction by Trizol gives different results (low quality) when compared to column based extraction method (Santiago-Vazquez, Ranzer, and Kerr, 2006). Furthermore, the use of kits containing phenol (such as Trizol ® or Qiazol ®) is risky.…”

Section: Introductionmentioning

confidence: 99%

Comparison of five RNA extraction methods from rabbit’s blood

Nour¹,

Barbour²,

Depint³

et al. 2010

ABJNA

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Extraction of RNA from eukaryotic cells is a crucial stage in many molecular biology studies. Thus it is essential to use the most appropriate method that maximizes the yield and maintains the integrity of extracted RNA. The present work compared five manual RNA extraction methods of different dilutions of rabbit blood. The assessment of the concentration and purity of the extracted RNA were used as parameters to evaluate the efficiency of each method. Statistical analysis showed that the most effective method for RNA extraction from rabbit blood is by following the protocol of the RNeasy Lipid Tissue kit, but it requires the use of toxic reagents. The most efficient kit using no toxic reagents is the RNeasy protect animal blood.

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“…Minogue et al [61] Rat NP, AF and AC tissues Modified TRIspin method Lee et al [128] In this study, we chose to use TRIzol for the RNA extraction as some studies have suggested the yield of RNA obtained using the guanidine isothiocyanate (GITC) based method can be higher than the column based method for some samples [129][130][131][132]. The studies in Table 3-2 either used TRIzol extraction or TRIspin method for RNA isolation.…”

Section: Tissue Extraction Referencementioning

confidence: 99%

“…RNeasy) are two commonly used methods for RNA extraction. Some studies have suggested the yield of RNA obtained using the GITC-based method can be higher than the column based method for some samples [129][130][131][132]. Thus we used GITC based TRIzol to lyse the cells.…”

Section: Comparison Of Rna From Propanol Precipitation and Column Basmentioning

confidence: 99%

“…In some situations, only a small number of cells is available for obtaining RNA such as cells sorted using fluorescent assisted cell sorting (FACS) [152], from tissues with a low cell density such as cartilage [153] and intervertebral disc [26] and when numerous cell culture conditions are tested with limited cell source. Some studies have suggested the yield of RNA obtained using the guanidine isothiocyanate (GITC) based method is higher than the column based method for some samples [129][130][131][132]. Besides, TRIzol (a GITC based reagent) allows the simultaneous extraction of RNA, DNA and protein from the same sample [154].…”

Section: Cryosection the Tissues And Lyse The Cells In Trizolmentioning

confidence: 99%

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Technique and method development for intervertebral disc (IVD) research

Lee¹,

李芷恩²

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Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. The direct and indirect cost of managing back pain posts heavy socioeconomic burden to the society. Improved technologies and techniques together with well documented experiments will benefit the research field by saving effort in doing the optimization in every laboratory and avoiding experiment failure due to incomplete understanding of the procedures. These can accelerate scientific discovery, reduce the sacrifice of animals and enable a more effective use of funding.Nucleus pulposus (NP) is the central part of IVD. Differences in matrix compositions in human NP clinical samples demand different cell isolation protocols for optimal results but there is no clear guide about this to date. Sub-optimal protocols may result in low cell yield, limited reliability of results or even failure of experiments. We experimented different isolation protocols to study different parameters involved and suggested some rules for cell isolation in three main applications: RNA extraction for phenotyping, cell isolation for cell culture, and characterization by flow cytometry.In addition, instead of extracting RNA from isolated cells, extraction of RNA from tissues directly may avoid the change of RNA levels during the cell isolation process.However, extraction of RNA directly from human and large animal IVD tissue is technically challenging due to its tough nature, low cell-to-matrix ratio and high proteoglycan content. Thus we developed a method for RNA extraction from bovine disc tissues by integrating the use of cryosectioning, additional phase separation and high salt precipitation into conventional guanidinium thiocyanate based method. With this method, RNA could be extracted from the NP tissue directly but the concentration was low. A shift toward 270 nm was observed in its UV spectrum which was due to phenol contamination. This caused an overestimation of RNA concentration. Hence we developed a computational method based on UV spectra for correcting the In short, different methods related to IVD research were developed and optimized. With improved methods together with a better understanding of the underlying rationale, researchers can save time and cost in their experiments and reduce experiment failure rate. This will help to accelerate researches. New methods also enable studies which were not feasible in the past.(484 words)

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Comparison of two total RNA extraction protocols using the marine gorgonian coral Pseudopterogorgia elisabethae and its symbiont Symbiodinium sp

Cited by 26 publications

References 8 publications

Extraction of RNA from tough tissues with high proteoglycan content by cryosection, second phase separation and high salt precipitation

Extraction of RNA from tough tissues with high proteoglycan content by cryosection, second phase separation and high salt precipitation

Comparison of five RNA extraction methods from rabbit’s blood

Technique and method development for intervertebral disc (IVD) research

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