Comparison of Different Methods of RNA Preparation from Peripheral Blood for Nucleic Acid Amplification Assay

Lee, Dong‐Eun; Lee, Hanna; Lee, Seon Duk; Han, Hyung Soo; Choe, Jae Young; Yun, Sora; Park, Hong-Jun; Park, Jung-Bae; Kim, Jong Kun

doi:10.4103/ijmm.ijmm_18_104

Cited by 3 publications

(5 citation statements)

References 9 publications

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“…Several reports evaluating different extraction methodologies concluded that RNA yield contributes significantly to technical variation across methods [11,12,14,25,29]. We reported that RNA content in human blood ranges from 6-22 μg / ml [7], reaching concentrations greater than previously reported in the literature [3][4][5][6][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26].…”

Section: Introductionmentioning

confidence: 72%

“…A variety of methodologies are available for the extraction and recovery of RNA from whole blood. In addition, novel collection tubes, such as PAXgene and Tempus tubes, also can be employed for RNA stabilization [3,4,6,8,[12][13][14][15][16][17][18][19][20][21][23][24][25][26]. Although published microarray and RNA sequencing studies consistently use "good quality" RNA that is defined by A260/280, A260/230 ratios and RIN values, differences in gene expression are frequently reported and findings are difficult to replicate across various experimental platforms.…”

Section: Discussionmentioning

confidence: 99%

“…Based on an average blood concentration of 14.58 μg of RNA / ml of blood, sequencing 1 μg of RNA from the C35 samples constitutes ~7% of the RNA in the sample. Previously reported column-based RNA yields for human blood are much lower ranging 1-8 μg of RNA / ml of blood [3,4,6,8,[12][13][14][15][16][17][18][19][20][21][23][24][25][26]. Using 1 μg of RNA from these extractions would constitute 12-100% of the RNA from the sample.…”

Section: E)mentioning

confidence: 98%

“…Each of the various blood stabilization tubes have unique proprietary ingredients designed to stabilize the nucleic acids. The extraction procedures routinely employed to purify and recovery RNA from blood samples add additional variability since they employ different extraction technologies such as: phenol-based extractions [3-11, 13, 22], silica gel column purification procedures [3][4][5][6][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26], glass fiber extraction columns [26], magnetic bead extractions [16-17, 19, 23] and assorted blood cell enrichment methodologies coupled with various extraction protocols [6,[8][9][10][11]17]. The total quantity of RNA recovered from whole blood differs significantly between these various extraction methodologies , but the reported RNA purity (A 260/280 > 1.9 and A 260/230 ratios > 1.7) and integrity-based RIN values (RIN > 7) are in the acceptable range for microarray and RNA sequencing studies [27,28].…”

Section: Introductionmentioning

confidence: 99%

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Whole blood RNA extraction efficiency contributes to variability in RNA sequencing data sets

Wilfinger,

Eghbalnia,

Mackey

et al. 2023

PLoS ONE

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Numerous methodologies are used for blood RNA extraction, and large quantitative differences in recovered RNA content are reported. We evaluated three archived data sets to determine how extraction methodologies might influence mRNA and lncRNA sequencing results. The total quantity of RNA recovered /ml of blood affects RNA sequencing by impacting the recovery of weakly expressed mRNA, and lncRNA transcripts. Transcript expression (TPM counts) plotted in relation to transcript size (base pairs, bp) revealed a 30% loss of short to midsized transcripts in some data sets. Quantitative recovery of RNA is of considerable importance, and it should be viewed more judiciously. Transcripts common to the three data sets were subsequently normalized and transcript mean TPM counts and TPM count coefficient of variation (CV) were plotted in relation to increasing transcript size. Regression analysis of mean TPM counts versus transcript size revealed negative slopes in two of the three data sets suggesting a reduction of TPM transcript counts with increasing transcript size. In the third data set, the regression slope line of mRNA transcript TPM counts approximates zero and TPM counts increased in proportion to transcript size over a range of 200 to 30,000 bp. Similarly, transcript TPM count CV values also were uniformly distributed over the range of transcript sizes. In the other data sets, the regression CV slopes increased in relation to transcript size. The recovery of weakly expressed and /or short to midsized mRNA and lncRNA transcripts varies with different RNA extraction methodologies thereby altering the fundamental sequencing relationship between transcript size and TPM counts. Our analysis identifies differences in RNA sequencing results that are dependent upon the quantity of total RNA recovery from whole blood. We propose that incomplete RNA extraction directly impacts the recovery of mRNA and lncRNA transcripts from human blood and speculate these differences contribute to the “batch” effects commonly identified between sequencing results from different archived data sets.

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Section: Introductionmentioning

confidence: 72%

Section: Discussionmentioning

confidence: 99%

Section: E)mentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Whole blood RNA extraction efficiency contributes to variability in RNA sequencing data sets

Wilfinger,

Eghbalnia,

Mackey

et al. 2023

PLoS ONE

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“…Hal tersebut dikarenakan bukan hanya DNA saja yang mampu menyerap sinar UV pada 260 nm, tetapi RNA juga memiliki daya serap yang besar pada panjang gelombang 260 nm. Keberadaan RNA bisa disebut sebagai kontaminan jika berada dalam larutan DNA (Lee et al, 2018). Oleh karena itu, proses purifikasi menjadi penting agar hasil ekstraksi yang didapat menjadi lebih murni.…”

Section: Pembahasanunclassified

Concentration and purity of DNA extraction with sonication and spin column methods from the sputum sample of tuberculosis patient

Saputra,

Indra,

Djuminar

et al. 2024

curr biomed

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Background: The Polymerase Chain Reaction (PCR) method can identify Mycobacterium tuberculosis in a sputum sample of a patient with TB (TB). One crucial step to ensure accurate PCR results is the DNA extraction process. Objective: The research aims to compare the concentration and purity of DNA from the sputum of TB patients using ultrasound and spin column extraction techniques. Methods: The research uses descriptive study designs with post-only design strategies. The primary data was derived from 18 sputum specimens from TB patients. Concentration measurement and DNA purity testing using a nanodrop spectroscopic photometer. Results: DNA extraction by ultrasound method has an average concentration of 18.9 ± 8.5 ng/L, with a peak of 37.6 ng/ L. The spin column method produces an average of 55.5 ± 27.9 ng/μL; the peak is 105.0 ng/ μL. The purity value of the DNA extract is in the range of 1.8 ± 2.0 with the ultrasound method of 61% and the spin column of 78%. Conclusion: The sonication method has a lower average concentration and a higher percentage of purity than the spin column method, and there are differences in concentrations and purity values between the two methods.

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A simple blood preparation method for nucleic acid amplification tests using membranes

Kim

Lee

et al. 2019

THC

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Comparison of Different Methods of RNA Preparation from Peripheral Blood for Nucleic Acid Amplification Assay

Abstract: These results demonstrate that despite the lower purity and integrity of RNA, our RNA preparation protocol is simple and rapid and shows reasonable performance in RT-LAMP.

Cited by 3 publications

References 9 publications

Whole blood RNA extraction efficiency contributes to variability in RNA sequencing data sets

Whole blood RNA extraction efficiency contributes to variability in RNA sequencing data sets

Concentration and purity of DNA extraction with sonication and spin column methods from the sputum sample of tuberculosis patient

A simple blood preparation method for nucleic acid amplification tests using membranes

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