Methodology An alternate method for DNA and RNA extraction from clotted blood

Zakaria, Zalina; Umi, Shakina Haridan; Mokhtar, Smail; Mokhtar, U; Zaiharina, M.Z.; Aziz, Athar; Hoh, Boon Peng

doi:10.4238/2013.february.4.4

Cited by 20 publications

(20 citation statements)

References 18 publications

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“…Our results showed a significant increase of DNA extraction in modified salting‐out method and modified QIAamp® DNA Blood Midi Kit when compared with QIAamp ® DNA Blood Midi Kit ( P < 0.001, 75.43 ± 47.12 and 91.48 ± 63.07 vs 42.46 ± 26.21), suggesting that the quantity of DNA was higher when a mechanical device was used. This observation is consistent with previous results which indicated a mechanical breakage of the clots is useful for enhancing the amount of DNA …”

Section: Discussionsupporting

confidence: 94%

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A method for improving the efficiency of DNA extraction from clotted blood samples

Mardan-Nik

Soflaei

Biabangard‐Zak

et al. 2019

Clinical Laboratory Analysis

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BackgroundThe efficient and rapid extraction of high‐quality genomic DNA from clotted blood samples, which normally have a low yield and poor quality, is an important factor in genomic research. The objective of this study was to develop a simple and safe technique for dispersing the blood clots by the ball bearing metal shots. Normally, such clot samples may not have an acceptable yield by conventional DNA extraction methods. Also, in the present study, we have further investigated to improve salting‐out DNA extraction methods.MethodsInitially, 500 µL phosphate‐buffered saline (PBS) (1×) and two ball bearing metal shots were added to each tube of the clotted blood sample and then were gently rotated in an electric laboratory rotator for 1 hour at room temperature (18‐25°C). Genomic DNA was then extracted from samples using a modified salting‐out method and a modified QIAamp® DNA Blood Midi Kit and was compared with QIAamp® DNA Blood Midi Kit as a control. An assessment of the concentration and quality of the extracted DNA was performed using the UV‐visible spectrophotometer. The isolated DNA proved amenable to PCR amplification and gel electrophoresis.ResultsThe yield and purity of DNA obtained by these three methods were significantly different (P < 0.001), with a higher yield in the modified salting‐out method.ConclusionsOur proposed modified salting‐out method is simple and efficient for the isolation of DNA from old blood clot samples. It is both easy to use and is of low cost in routine laboratory tasks.

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Section: Discussionsupporting

confidence: 94%

“…The clot remaining from blood samples collected for serum chemistry measurements is also a great DNA source for genetic or forensic studies. Some methods have optimized DNA extraction from clotted blood, which are normally discarded …”

Section: Introductionmentioning

confidence: 99%

A method for improving the efficiency of DNA extraction from clotted blood samples

Mardan-Nik

Soflaei

Biabangard‐Zak

et al. 2019

Clinical Laboratory Analysis

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“…Genomic DNA was extracted either from whole, or clotted blood using commercially available kit, with slight modification, according to Zuraihan et al [15].…”

Section: Genotypingmentioning

confidence: 99%

Common variants of chemokine receptor gene CXCR3 and its ligands CXCL10 and CXCL11 associated with vascular permeability of dengue infection in peninsular Malaysia

et al. 2015

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Dengue causes significantly more human disease than any other arboviruses. It causes a spectrum of illness, ranging from mild self-limited fever, to severe and fatal dengue hemorrhagic fever, as evidenced by vascular leakage and multifactorial hemostatic abnormalities. There is no specific treatment available till date. Evidence shows that chemokines CXCL10, CXCL11 and their receptor CXCR3 are involved in severity of dengue, but their genetic association with the susceptibility of vascular leakage during dengue infection has not been reported. We genotyped 14 common variants of these candidate genes in 176 patients infected with dengue. rs4859584 and rs8878 (CXCL10) were significantly associated with vascular permeability of dengue infection (P<0.05); while variants of CXCL11 showed moderate significance of association (P=0.0527). Haplotype blocks were constructed for genes CXCL10 and CXCL11 (5 and 7 common variants respectively). Haplotype association tests performed revealed that, "CCCCA" of gene CXCL10 and "AGTTTAC" of CXCL11 were found to be significantly associated with vascular leakage (P=0.0154 and 0.0366 respectively). In summary, our association study further strengthens the evidence of the involvement of CXCL10 and CXCL11 in the pathogenesis of dengue infection.

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“…Good quality RNA is important in modern molecular biological methods. [14,15] Besides, dealing with a gene source like animal tissues as an example (leeches source of hirudin as in this study) requires a special treatment as immediate freezing of fresh tissue samples and the use liquid nitrogen. [16] Because of that scientists tried to produce a gene without DNA or RNA extraction, especially if the gene in question is not large therefore the method known as overlap extension PCR was introduced.…”

Section: Discussionmentioning

confidence: 99%

Production and purification of constructed recombinant hirudin in BL21(DE3) strain

Al-Badran

Al-Fadal

2017

JBEI

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Background/Objective: Hirudin, an extract from the leech, has powerful antithrombin activity affecting the blood coagulation pathway, it is the most potent natural inhibitor of thrombin, it binds thrombin with high affinity, so, the aim of this study was to build hirudin gene by overlapping extension PCR then cloning and expression in BL21(DE3) strain. Methods: Hirudin gene constructed with four modified primers then the final product amplified by two primers named as A, B by using overlapping extension PCR, for gene expression, BL21(DE3) strain was used under the control of T7 promoter in pET-16b vector and for hirudin production, LB broth medium was used as fermentation medium, to detect the expression of hirudin in BL21(DE3) strain in fermentation media real-time PCR was used. Hirudin protein purified at first by (Immobilized metal affinity chromatography) IMAC, then this protein was dialysis and treated with factor Xa to eliminate His-tag. Then hirudin purified by DEAE sepharose and SP sepharose column, the concentration of protein determined by ELISA, furthermore the activity evaluated by thrombin titration and activated partial thromboplastin time (APPT) test. Results: Hirudin gene constructed in two round PCR first round produced two products (product 1,117 bp while product 2,114 bp) the second-round PCR gave the final product 213 bp. The resulted band from gel electrophoresis for constructed vector pET-16b-HirudinS was (5,901 bp). Hirudin expression was established by real-time PCR with Ct value (20.28). The analysis on 15% SDS-PAGE for the SP sepharose column illustrated the hirudinS protein band with size about ∼10.8. Concentration of produced hirudin within its solution reached to 1.75 ng, thrombin titration method showed that the hirudin protein required 300 µl from thrombin to clot, also, APPT test showed that hirudin elongated clotting time to 7 min in comparison with 6min for aspirin and the statistical analysis results for APPT test illustrated that there was no significant difference between hirudin and aspirin. Conclusion: This study approved that overlapping extension PCR is a good strategy for building hirudin gene and it's successfully expressed in BL21(DE3) strain.

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Methodology An alternate method for DNA and RNA extraction from clotted blood

Cited by 20 publications

References 18 publications

A method for improving the efficiency of DNA extraction from clotted blood samples

A method for improving the efficiency of DNA extraction from clotted blood samples

Common variants of chemokine receptor gene CXCR3 and its ligands CXCL10 and CXCL11 associated with vascular permeability of dengue infection in peninsular Malaysia

Production and purification of constructed recombinant hirudin in BL21(DE3) strain

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