scholarly journals Selection of RNA isolation method for molecular detection of grapevine viruses

2017 ◽  
Vol 38 (SI 2 - 6th Conf EFPP 2002) ◽  
pp. 267-270
Author(s):  
P. Komínek
Keyword(s):  
Extraction Method ◽  
Molecular Detection ◽  
Rna Isolation ◽  
Rt Pcr ◽  
Isolation Method ◽  
Phloem Tissue ◽  
Total Rna ◽  
Chloroform Extraction ◽  
Selection Of

Grapevines infected with Grapevine leafroll-associated virus-1 (GLRaV-1) and Grapevine leafroll-associated virus-3 (GLRaV-3) were selected. Total RNA was isolated from grapevine phloem tissue scrapped from dormant canes by three different methods: extraction with urea buffer followed with phenol-chloroform extraction, method using Concert<sup>TM</sup> reagent (Invitrogen) followed with chloroform-isopropylalcohol extraction, and procedure using RNeasy Plant Mini Kit (Qiagen). The highest yield of RNA was obtained using Concert<sup>TM</sup> reagent. If this RNA was used in RT-PCR, GLRaV-1 and GLRaV-3 were easily detected. From RNA isolated by other two methods these viruses were not detected.

New Data on Robustness of Gene Expression Signatures in Leukemia: Comparison of Three Distinct Total RNA Preparation Procedures.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4288-4288
Author(s):  
Marta Campo ◽  
Andrea Zangrando ◽  
Luca Trentin ◽  
Rui Li ◽  
Wei-min Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Data ◽  
Rna Isolation ◽  
Expression Data ◽  
Microarray Technology ◽  
Isolation Method ◽  
Acute Leukemias ◽  
Preparation Methods ◽  
Total Rna ◽  
Total Rna Isolation

Abstract Gene expression microarrays had been used to classify known tumor types and various hematological malignancies (Yeoh et al, Cancer Cell 2002; Kohlmann et al, Genes Chromosomes Cancer 2003), enforcing the objective that microarray analysis could be introduced soon in the routine classification of cancer (Haferlach et al, Blood 2005). However, there’re still doubts about gene expression experiments performance in clinical laboratory diagnosis. For instance, the quality of starting material is a major concern in microarray technology and there are no data on the variation in gene expression profiles ensuing from different RNA extraction procedures. Here, as part of the internal multicenter MILE Study program, we assess the impact of different RNA preparation methods on gene expression data, analyzing 27 patients representative of nine different subtypes of pediatric acute leukemias. We compared the three currently most used protocols to isolate RNA for routine diagnosis (PCR assays) and microarray experiments. They are named as method A: lysis of mononuclear leukemia cells, followed by lysate homogeniziation, followed by total RNA isolation; method B: TRIzol RNA isolation, and method C: TRIzol RNA isolation followed by total RNA purification step. The methods were analyzed in triplicates for each sample (24) and additional three samples were performed in technical replicates of three data sets for each preparation (HG-U133 Plus 2.0). Method A results in better total RNA quality as demonstrated by 3′/5′ GAPD ratios and by RNA degradation plots. High comparability of gene expression data is found between samples in the same leukemia subclasses and collected with different RNA preparation methods thus demonstrating that sample preparation procedures do not impair the overall signal distribution. Unsupervised analyses showed clustering of samples first by each patient’s replicate conditions, then by leukemia type, and finally by leukemia lineage. In fact, B-ALL samples are clustered together, separately from T-ALL and AML, demonstrating that clustering reflects biological differences between leukemias and that the RNA isolation method is a secondary effect. Also, supervised cluster analyses highlight that samples are grouped depending on intra-lineage features (i.e. chromosomal aberrations) thus confirming the clustering organizations as reported in recent gene expression profiling studies of acute leukemias. Our study shows that biological features of pediatric acute leukemia classes largely exceed the variations between different total RNA sample preparation protocols. However, technical replicates analyses reveal that gene expression data from method A have the lowest degree of variation, are more reproducible and more precise as compared to the other two methods. Furthermore, compared to methods B and C, method A produces more differentially expressed probe sets between distinct leukemia classes and is therefore considered the more robust RNA isolation procedure for gene expression experiments using high-density microarray technology. We therefore conclude that method A (initial homogenization of the leukemia cell lysate followed by total RNA isolation) combined with a standardized microarray analysis protocol is highly reproducible and contributes to robustness of gene expression data and that this procedure is most practical for a routine laboratory use.

Total RNA‐Isolation of Abdominal Hernia of Rats for Quantitative Real‐Time Reverse Transcription (RT) PCR Assays

2007 ◽  
Vol 38 (1) ◽  
pp. 87-93
Author(s):  
Christian Morsczeck ◽  
Michael Korenkov ◽  
Manfred Nagelschmidt ◽  
Domonkos Feher ◽  
Jörg Michael Schierholz
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Rna Isolation ◽  
Abdominal Hernia ◽  
Rt Pcr ◽  
Total Rna ◽  
Pcr Assays ◽  
Total Rna Isolation

scholarly journals Isolation and Detection of Enterovirus RNA from Large-Volume Water Samples by Using the NucliSens miniMAG System and Real-Time Nucleic Acid Sequence-Based Amplification

2005 ◽  
Vol 71 (7) ◽  
pp. 3734-3740 ◽  
Author(s):  
Saskia A. Rutjes ◽  
Ronald Italiaander ◽  
Harold H. J. L. van den Berg ◽  
Willemijn J. Lodder ◽  
Ana Maria de Roda Husman
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Large Volume ◽  
Water Samples ◽  
Extraction Method ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Rt Pcr ◽  
Virus Rna ◽  
Nasba Assay

ABSTRACT Concentration of water samples is a prerequisite for the detection of the low virus levels that are present in water and may present a public health hazard. The aim of this study was to develop a rapid, standardized molecular method for the detection of enteroviruses in large-volume surface water samples, using a concentration method suitable for the detection of infectious viruses as well as virus RNA. Concentration of water was achieved by a conventional filter adsorption-elution method and ultrafiltration, resulting in a 10,000-fold concentration of the sample. Isolation of virus RNA by a silica-based RNA extraction method was compared with the nonmagnetic and magnetic NucliSens RNA isolation methods. By using the silica-based RNA extraction method in two out of five samples, enterovirus RNA was detected, whereas four out of five samples were positive following RNA isolation with magnetic silica beads. Moreover, estimated RNA levels increased at least 100 to 500 times. Furthermore, we compared enterovirus detection by an in-house reverse transcription (RT)-PCR with a novel commercially available real-time nucleic acid sequence-based amplification (NASBA) assay. We found that the rapid real-time NASBA assay was slightly less sensitive than our in-house RT-PCR. The advantages, however, of a commercial real-time NASBA assay, like the presence of an internal control RNA, standardization, and enormous decrease in turnaround time, makes it an attractive alternative to RT-PCR.

scholarly journals COMPARISON OF TWO TOTAL RNA EXTRACTION PROTOCOLS FROM CHO-K1 CELLS FOR RT-PCR: CUT-OFF COST FOR RESEARCHERS

2014 ◽  
Vol 15 (1) ◽  
Author(s):  
Vasila Packeer Mohamed ◽  
Yumi Z. H-Y. Hashim ◽  
A. Amid ◽  
M. Mel
Keyword(s):  
Mammalian Cells ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Primary Concern ◽  
Rt Pcr ◽  
Total Rna ◽  
Rna Purification ◽  
Dnase Treatment ◽  
Study Gene Expression ◽  
Significant Difference

ABSTRACT: Various methods have been described to extract RNA from adherent mammalian cells. RNA isolation in conjunction with reverse transcription polymerase chain reaction (RT-PCR) is a valuable tool used to study gene expression profiling. This approach is now being used in mammalian cell bioprocessing to help understand and improve the system. The objective of this study was to compare and determine the most suitable RNA extraction method for CHO-K1 cells in a setting where a relatively large amount of samples was involved. Total RNA was extracted using Total RNA purification kit (without DNase treatment; Norgen, Canada) and RNeasy mini kit (with DNase treatment; Qiagen, USA) respectively. The extracted RNA was then reverse transcribed, and the cDNA was subjected to PCR-amplifying 18S. Yield from RNeasy kit was significantly higher (0.316 ± 0.033 µg/µl; p=0.004) than Total RNA purification kit (0.177 ± 0.0243 µg/µl). However, RNA purity for both methods was close to 2.0 and there was no significant difference between the methods. Total RNA purification kit is less expensive than RNeasy kit. Since there is no DNase treatment step in the former, extraction time for RNA is shorter. When the extracted RNA was subjected to RT-PCR, both methods were able to show detection of 18S at 219 bp.   Therefore, this study demonstrates that both protocols are suitable for RNA extraction for CHO-K1 cells. RNeasy mini kit (Qiagen) is recommended if higher yields is the primary concern and Total RNA Purification kit (Norgen) is recommended if time and cost are concerned. ABSTRAK: Pelbagai kaedah telah digunakan untuk mengekstrak RNA daripada sel mamalia lekat.  Pemencilan RNA dengan menggunakan reaksi rantai polimerase transkripsi berbalik (RT-PCR) merupakan kaedah penting yang digunakan dalam mengkaji pernyataan gen berprofil.  Pendekatan ini kini digunakan dalam pemprosesan bio sel mamalia untuk memahami dan menambah baik sistem.  Tujuan kajian dijalankan adalah untuk menentukan dan membandingkan kaedah ekstraksi RNA yang paling sesuai bagi sel CHO-K1 di persekitaran di mana kadar sampel yang agak besar terlibat. Jumlah RNA  diekstrak menggunakan kit penulenan Jumlah RNA (tanpa rawatan DNase; Norgen, Canada) dan kit mini RNeasy (dengan rawatan DNase; Qiagen, USA).  RNA yang diekstrak kemudiannya diterbalikkan transkripsi, dan cDNA menjalani penguat PCR 18S. Hasil daripada kit RNeasy adalah lebih tinggi (0.316 ± 0.033 µg/µl; p=0.004) berbanding dengan kit penulenan Jumlah RNA (0.177 ± 0.0243 µg/µl). Walaupun begitu, kaedah penulenan RNA untuk kedua-duanya hampir 2.0 dan tidak terdapat perbezaan yang ketara antara keduanya. Kit penulenan Jumlah RNA adalah lebih murah berbanding dengan kit RNeasy. Memandangkan tidak ada langkah rawatan DNase dengan penggunaan kit Jumlah RNA, tempoh ekstrak RNA nya lebih pendek. Apabila RNA yang telah diekstrak menjalani RT-PCR, kedua-dua kaedah berjaya mengesan 18S pada 219 bp.   Kesimpulannya, kajian ini menunjukkan kedua-dua kaedah sesuai untuk mengekstrak RNA bagi sel CHO-K1. Kit mini RNeasy (Qiagen) lebih sesuai jika hasil yang tinggi diinginkan dan kit penulenan Jumlah RNA (Norgen) pula ideal, jika kos dan masa berkepentingan.

scholarly journals First Report of Tobacco rattle virus Causing Corky Ringspot in Potatoes Grown in Minnesota and Wisconsin

Plant Disease ◽  
2008 ◽  
Vol 92 (8) ◽  
pp. 1254-1254 ◽  
Author(s):  
N. C. Gudmestad ◽  
I. Mallik ◽  
J. S. Pasche ◽  
J. M. Crosslin
Keyword(s):  
Nucleotide Sequence ◽  
Sequence Data ◽  
Potato Crop ◽  
Tobacco Rattle Virus ◽  
Rna Isolation ◽  
Rt Pcr ◽  
Total Rna ◽  
First Report ◽  
Tuber Necrosis ◽  
Corky Ringspot

In July 2007, potato tubers cv. Russet Burbank (RB) with necrotic arcs and spots were detected in three fields in Buffalo County, Wisconsin and one field in Benson County, Minnesota. Umatilla Russet (UR) potatoes harvested from the west half of a field in Swift County, MN had similar, but visually distinct necrotic lesions. Portions of one field in Minnesota were abandoned, and the stored potato crop from two fields in Wisconsin was rejected by processors, representing a total crop loss due to tuber necrosis. Tuber symptoms displayed in both cultivars resembled those described for corky ringspot caused by Tobacco rattle virus (TRV) (4). Total RNA was isolated from necrotic tuber tissue crushed in liquid nitrogen and extracted using the Total RNA Isolation Kit (Promega Corp., Madison, WI). These extracts were tested for the presence of TRV by reverse transcription (RT)-PCR using primers complementary to nucleotides 6555 to 6575 and identical to nucleotides 6113 to 6132 within the 3′ terminal open reading frame of TRV RNA-1 (3). The expected 463-bp fragments were amplified from RB tubers. Nucleotide sequences from a Wisconsin and Minnesota isolate (GenBank Accession Nos. EU569290 and EU569291, respectively) were 99 to 100% identical to the corresponding region in a published TRV sequence (GenBank Accession No. AF055912). A 396-bp fragment was amplified from UR tubers and sequence data (GenBank Accession No. EU569292) indicated a unique 63 nucleotide sequence was substituted for a 129 nucleotide sequence spanning residues 227 to 357 of the 463-bp amplicon from the RB TRV isolates. Seven fragments were sequenced from different UR tubers and the 396-bp fragment was identical among them. The sequence outside the substituted region had 92% identity to the published TRV sequence. Amplification of the full-length TRV RNA2 using primers 179/180 located in the 5′ and 3′ untranslated regions (2) was successful for 28 and 0% of the RB and UR samples, respectively, suggesting that the RNA2 is not present in these strains or has undergone significant mutation. TRV-infected sap from both potato cultivars was mechanically transmitted to tobacco cv. Samsun NN and these plants subsequently tested positive for TRV by ELISA using ATCC antiserum PVAS 820. Ninety tubers exhibiting mild to severe symptoms of TRV were planted in the greenhouse. Each tuber was bisected laterally; necrotic tissue was removed from one half of the tuber and tested for the presence of TRV using RT-PCR protocols described above for RNA1. The remaining half was bisected horizontally and both sections were planted. Foliage from each emerged plant was subsequently also tested by RT-PCR for TRV RNA1. All RB tubers from Wisconsin tested positive for TRV, but only 7 of 24 emerged plants tested positive. Only 72% of the UR tubers and 4 of 25 emerged plants tested positive. TRV has been confirmed in California, Colorado, Florida, Idaho, Michigan (1), Oregon, and Washington. To our knowledge, this is the first report of corky ringspot in potato caused by TRV in Minnesota and Wisconsin. References: (1) W. W. Kirk et al. Plant Dis. 92:485, 2008. (2) S. A. MacFarlane. J. Virol. Methods. 56:91, 1996. (3) D. J. Robinson. J. Virol. Methods 40:57, 1992. (4) S. A. Slack. Tobacco rattle virus. Page 71 in: Compendium of Potato Diseases. 2nd ed. W. R. Stevenson et al., eds. The American Phytopathological Society, St. Paul, MN, 2001.

Optimisation of total RNA isolation method from bird sperm

2013 ◽  
Vol 13 ◽  
pp. 29
Author(s):  
A. Partyka ◽  
A. Zielak-Steciwko ◽  
W. Niżański ◽  
J. Bajzert
Keyword(s):  
Rna Isolation ◽  
Isolation Method ◽  
Total Rna ◽  
Total Rna Isolation

Total RNA-Isolation of Abdominal Hernia of Rats for Quantitative Real-Time Reverse Transcription (RT) PCR Assays

2008 ◽  
Vol 38 (2) ◽  
pp. 129-135
Author(s):  
Christian Morsczeck ◽  
Michael Korenkov ◽  
Manfred Nagelschmidt ◽  
Domos Feher ◽  
Jörg Michael Schierholz
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Rna Isolation ◽  
Abdominal Hernia ◽  
Rt Pcr ◽  
Total Rna ◽  
Pcr Assays ◽  
Total Rna Isolation
Sign in / Sign up

Export Citation Format

Share Document