scholarly journals Evaluation of RNA isolation methods for microRNA quantification in a range of clinical biofluids

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Henk P. Roest ◽  
Jan N. M. IJzermans ◽  
Luc J. W. van der Laan
Keyword(s):  
Solid Phase ◽  
Rna Isolation ◽  
Cdna Synthesis ◽  
Chloroform Extraction ◽  
Inhibitory Compounds ◽  
Isolation Methods ◽  
Synthetic Mirnas ◽  
Mirna Detection ◽  
Endogenous Mirnas ◽  
Heparinase I

Abstract Background Extracellular microRNAs (miRNAs), released from cells into biofluids, have emerged as promising biomarkers for diagnostic and prognostic purposes. Several RNA isolation methods are available for the analysis of these cell-free miRNAs by RT-qPCR. Not all methods, however, are equally suitable for different biofluids. Here, we extracted total RNA from four very diverse biofluids: serum, urine, bile, and graft preservation fluid (perfusate). Four different protocols were used: a phenol-chloroform extraction and alcohol precipitation in combination with a precipitation carrier (QP) and three different column-based isolation methods, one with phenol-chloroform extraction (RN) and two without (NG and CU). For this range of clinical biofluid samples, we evaluated the potential of these different RNA isolation methods assessing recovery efficiency and the co-purification of RT-qPCR inhibiting compounds. Results Differences were observed between each of the RNA isolation methods in the recovery of cel-miR-39, a synthetic miRNA spiked in during the workup procedure, and for endogenous miRNAs. Co-purification of heparin, a known RT-qPCR inhibitor, was assessed using heparinase I during cDNA synthesis. RT-qPCR detection of synthetic miRNAs cel-miR-39, spiked in during RNA workup, cel-miR-54, spiked in during cDNA synthesis, and endogenous miRNAs was strongly improved in the presence of heparinase I for some, but not all, isolation methods. Other, co-isolated RT-qPCR inhibitors were not identified, except for biliverdin, which co-isolated from some bile samples with one of the methods. In addition, we observed that serum and urine contain compounds that enhance the binding of heparin to certain solid-phase columns. Conclusions For reliable measurements of miRNA-based biomarkers in biofluids, optimization of RNA isolation procedures is recommended as methods can differ in miRNA detection and in co-purification of RT-qPCR inhibitory compounds. Heparinase I treatment confirmed that heparin appeared to be the major RT-qPCR inhibiting compound, but also biliverdin, co-isolated from bile, could interfere with detection.

scholarly journals Evaluation of RNA isolation methods for microRNA quantification in a range of clinical biofluids.

2020 ◽  
Author(s):  
Henk Roest ◽  
Jan N.M. IJzermans ◽  
Luc. J.W. van der Laan
Keyword(s):  
Rna Isolation ◽  
Sample Type ◽  
Cdna Synthesis ◽  
Inhibitory Compounds ◽  
Isolation Methods ◽  
Synthetic Mirnas ◽  
Mirna Detection ◽  
Endogenous Mirnas ◽  
Heparinase I ◽  
Bile Samples

Abstract BACKGROUND Extracellular microRNAs (miRNAs), released from cells into biofluids, have emerged as promising biomarkers for diagnostic and prognostic purposes. For the analysis of these cell-free miRNAs in various biofluids by RT-qPCR, several RNA isolation methods are available. However, not all methods are equally suitable for different biofluids. The aim of this study is to evaluate the potential of different RNA isolation methods in a range of clinical biofluid samples.METHODS Total RNA was isolated from serum, urine, bile, and graft preservation fluid (perfusate) using four different protocols: phenol-chloroform extraction in combination with a precipitation carrier, and three different column-based isolation methods. Co-purification of heparin, a known RT-qPCR inhibitor, was assessed using heparinase I during cDNA synthesis. Synthetic miRNAs, spiked-in during RNA workup (cel-miR-39) or during cDNA synthesis (cel-miR-54), and endogenous miRNAs were quantified using RT-qPCR.RESULTS Recovery of cel-miR-39 significantly differed between methods with miRNeasy columns providing the best overall recovery in the four biofluids tested, as was also observed for endogenous miRNAs. Contamination of RNA with heparin differed between sample type and isolation method, and could be counteracted using heparinase I. Other co-isolated RT-qPCR inhibitors were not identified, except for biliverdin which co-isolated from some bile samples with one of the methods.CONCLUSIONS For reliable measurements of miRNA-based biomarkers in biofluids, optimization of RNA isolation procedures is recommended as methods can differ in miRNA detection and co-purification of RT-qPCR inhibitory compounds. Heparinase I treatment confirmed that heparin appeared to be the major RT-qPCR-inhibiting compound but also biliverdin, co-isolated from bile, could interfere with detection.

Spermatozoa input concentrations and RNA isolation methods on RNA yield and quality in bull (Bos taurus)

2015 ◽  
Vol 482 ◽  
pp. 32-39 ◽  
Author(s):  
Sivashanmugam Parthipan ◽  
Sellappan Selvaraju ◽  
Lakshminarayana Somashekar ◽  
Atul P. Kolte ◽  
Arunachalam Arangasamy ◽  
...  
Keyword(s):  
Bos Taurus ◽  
Rna Isolation ◽  
Yield And Quality ◽  
Isolation Methods

scholarly journals Konstruksi pustaka cDNA dari daun klon karet AVROS 2037 yang diinfeksi patogen Corynespora cassiicola Construction of a cDNA library from leaf of AVROS 2037 rubber clone infected by Corynespora cassiicola pathogen

2016 ◽  
Vol 73 (2) ◽  
Author(s):  
. NURHAIMI-HARIS ◽  
Hajrial ASWIDINNOOR ◽  
Antonius SUWANTO ◽  
Maggy T. SUHARTONO ◽  
Nurita TORUAN-MATHIUS ◽  
...  
Keyword(s):  
Cdna Library ◽  
Plasmid Vector ◽  
Rna Isolation ◽  
Corynespora Cassiicola ◽  
Resistant Clone ◽  
Cdna Library Construction ◽  
Cdna Synthesis ◽  
Cdna Insert ◽  
E Coli ◽  
Average Size

SummaryConstruction of cDNA library derived fromtranscripts made under certain condition is animportant first step to understand diseaseresistant mechanisms. To identify rubber genesor transcripts involved in defense responsetoward Corynespora cassiicola, cDNA librarywas constructed using rubber clone AVROS2037, one of resistant clone to this pathogen.cDNA library was constructed based on thestrategy of leaves infection using conidia, withthe assumption that transcript expression relatedto defense response would be induced bypathogen infection. RNA was isolated from leavesthree days after inoculation with conidia ofC. cassiicola. Steps involved in the cDNA libraryconstruction were RNA isolation, mRNApurification, cDNA synthesis, vector modifcation,cDNA insert ligation, plasmid transformation andclone verifications. Each gram of leaf producedapproximately 300  g RNA, and 0.25% of themwas mRNA. The mRNA was used to synthesizedcDNA. Ligation of cDNA and modified vectorwas facilitated by restriction enzyme SfiI. Theconstructs were transformed into the E. coliDH5 competent cells. A total of 8000 colonieswere produced. Random examination of 270colonies showed that approximately 93% of thesecolonies carried plasmid vector with DNA insertsize of 200 – 2000 bp, with average size of 500 –800 bp. cDNA library construction of rubberleaves from AVROS 2037 clone as well as somenecessary modification steps are presented in thispaper.RingkasanKonstruksi pustaka cDNA yang me-ngandung transkrip yang diekspresikan dalamkondisi tertentu merupakan tahap awal yangsangat penting dalam berbagai studi biologi.Untuk mengidentifikasi gen karet atau transkripyang berperan dalam respons pertahanan tanamankaret terhadap Corynespora cassiicola, pustakacDNA dibuat dengan menggunakan daun klonAVROS 2037 yang merupakan salah satu klonresisten terhadap patogen tersebut. PustakacDNA dibuat berdasarkan strategi menginfeksidaun dengan konidia C. cassiicola denganpertimbangan bahwa ekspresi transkrip yangberperan dalam respons pertahanan akandiinduksi oleh adanya infeksi patogen. Dengandemikian pustaka cDNA yang dibuat diharapkanmengandung gen atau bagian gen yang ber-hubungan dengan respons pertahanan. RNAdiisolasi dari daun setelah daun diinokulasiselama tiga hari dengan konidia C. cassiicola.Beberapa tahapan telah dilakukan, dimulaidengan isolasi RNA, pemurnian mRNA, sintesiscDNA, modifikasi vektor kloning, ligasi fragmencDNA utas ganda dengan vektor kloning sertatransformasi hasil ligasi ke bakteri Escherichiacoli DH5 kompeten. Dari setiap gram jaringandaun berhasil diisolasi RNA sekitar 300 g, dandari jumlah tersebut sekitar 0,25% mRNA dapatdiisolasi. mRNA yang diisolasi digunakan untuksintesis cDNA. cDNA dipotong dengan enzimrestriksi SfiI dan diligasi ke vektor plasmid yangdimodifikasi dengan menyisipkan situs enzimSfiI. cDNA-vektor rekombinan ditransformasi kedalam sel bakteri E. coli DH5 kompeten meng-gunakan metode standar. Transformasi konstrukini menghasilkan 8.000 koloni. Pengujian PCRterhadap 270 koloni yang dipilih secara acakmengindikasikan bahwa sekitar 93% kolonitersebut membawa cDNA sisipan dengan ukuranfragmen cDNA yang menyisip berkisar antara200 sampai 2000 bp. cDNA sisipan terbanyakterdapat pada ukuran antara 500 – 800 bp. Dalamtulisan ini dibahas tahap demi tahap proses yangdilakukan untuk membuat pustaka cDNA asaldaun karet klon AVROS 2037 serta beberapamodifikasi yang diperlukan.

scholarly journals Flowering gene expression in Indonesian long harvest black rice (Oryza sativa L. ‘Cempo Ireng’)

2019 ◽  
pp. 874-880 ◽  
Author(s):  
Yekti Asih Purwestri ◽  
Febri Adi Susanto ◽  
Anisa Nazera Fauzia
Keyword(s):  
Oryza Sativa L ◽  
Early Stage ◽  
Rna Isolation ◽  
Tropical Region ◽  
Black Rice ◽  
Cdna Synthesis ◽  
Human Diet ◽  
Flowering Genes ◽  
The Common ◽  
Great Potency

Many studies have reported the great potency of black rice as functional food for human diet. Cempo Ireng is one of Indonesian black rice cultivars with the highest content of anthocyanin. However, it also suffers from long harvest period. This experiment aims at investigating the behavior of the flowering genes in order to gain basic information to develop this cultivar. We sampled the leaves’ blades of Black Rice ‘Cempo Ireng’ at 48, 55, 68, 81, and 90 DAP then performed RNA isolation and cDNA synthesis, amplification of targeted flowering genes, and a semi-quantitative analysis to see the expression of flowering genes. Our results showed that the flowering genes Hd3a and RFT1 were redundantly up-regulated to induce flowering in black rice Cempo Ireng under a neutral day condition in a tropical region. We also noted that the patterns of FT-like genes and flowering regulatory genes including FT-L5, FT-L6, FT-L9, FT-L10 and Hd1, OsCOL4 were expressed together with two major flowering genes. FT-like genes were temporally co-expressed with two flowering genes Hd3a and RFT1, whereas the Hd1 had a unique expression pattern. Meanwhile, OsCOL4 as the flowering repressor was only detected in the early stage when the flowering gene Hd3a began to express. The results suggest that black rice Cempo Ireng has similar and conserved flowering pathway under a neutral day condition as indicated in the common rice flowering models.

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