Total RNA Extraction from Saccharomyces cerevisiae Using Hot Acid Phenol

2021 ◽  
Vol 2021 (12) ◽  
pp. pdb.prot101691
Author(s):  
Michael R. Green ◽  
Joseph Sambrook
Keyword(s):  
Blot Hybridization ◽  
Rna Extraction ◽  
Sodium Dodecyl ◽  
Northern Blot Hybridization ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Low Salt ◽  
Rigid Cell Wall ◽  
Polymerase Chain

Isolation of RNA from yeast is complicated by the need to first break the thick, rigid cell wall. The protocol provided here uses a cycle of heating and freezing of cells in the presence of phenol and the detergent sodium dodecyl sulfate (SDS). The extraction is performed in the presence of low salt so that, following separation of the aqueous and phenol phases by centrifugation, DNA can be collected from the interface while RNA remains in the aqueous phase. This protocol should yield ∼50–250 µg of RNA from 10 mL of culture. The RNA isolated using this approach is suitable for most follow-up applications such as northern blot hybridization, reverse transcriptase-polymerase chain reaction (RT-PCR), and cDNA construction.

scholarly journals Detection and Differentiation of Bovine Group A Rotavirus Serotypes using Polymerase Chain Reaction-Generated Probes to the VP7 Gene

1992 ◽  
Vol 4 (2) ◽  
pp. 148-158 ◽  
Author(s):  
Anil V. Parwani ◽  
Blair I. Rosen ◽  
Jorge Flores ◽  
Malcolm A. McCrae ◽  
Mario Gorziglia ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Blot Hybridization ◽  
Northern Blot Hybridization ◽  
Chain Reaction ◽  
Diarrhea Virus ◽  
Bovine Rotavirus ◽  
Group A ◽  
Polymerase Chain ◽  
Vp7 Gene ◽  
Hybridization Assays

Dot and Northern blot hybridization assays were developed to detect and differentiate group A bovine rotavirus serotypes using radiolabeled serotype 6 (Nebraska calf diarrhea virus [NCDV] and United Kingdom [UK] strains) or serotype 10 (Cracker [Cr] strain) VP7 gene probes. Partial length VP7-specific cDNA encompassing areas of major sequence diversity were generated by the polymerase chain reaction (PCR) using either cloned VP7 genes (NCDV and UK strains) or reverse transcribed mRNA (Cr strain) as templates. Radiolabeled probes prepared from the PCR-generated cDNA were tested at various stringency conditions to optimize the hybridization assays. At high stringency conditions (52 C, 50% formamide, 5 x standard saline citrate), the NCDV, UK, and Cr probes serotypically differentiated bovine rotavirus isolates in RNA samples prepared from cell culture-propagated viruses or in fecal specimens from infected gnotobiotic calves. The sensitivity and specificity of NCDV and Cr VP7 probes were characterized in dot blot hybridization assays, and the probes were estimated to detect at least 1 ng of viral RNA. The serotyping results obtained using VP7 probes were similar to those obtained using serologic assays. Further development of these assays may provide a useful means for the rapid detection and differentiation of bovine rotavirus serotypes in fecal samples from calves in the field.

scholarly journals Autologous Bone Marrow Transplantation for Acute Promyelocytic Leukemia in Second Remission: Prognostic Relevance of Pretransplant Minimal Residual Disease Assessment by Reverse-Transcription Polymerase Chain Reaction of the PML/RARα Fusion Gene

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 1321-1325 ◽  
Author(s):  
Giovanna Meloni ◽  
Daniela Diverio ◽  
Marco Vignetti ◽  
Giuseppe Avvisati ◽  
Saveria Capria ◽  
...  
Keyword(s):  
Median Time ◽  
Fusion Gene ◽  
Autologous Bone Marrow Transplantation ◽  
Autologous Bone Marrow ◽  
Promyelocytic Leukemia ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Autologous Bone

Abstract Reverse-transcription polymerase chain reaction (RT-PCR) of the PML/RARα fusion gene may predict relapse in acute promyelocytic leukemia (APL) patients in hematologic complete remission (CR). We have prospectively studied by RT-PCR 15 PML/RARα+ APL patients undergoing autologous bone marrow transplantation (ABMT) in second CR. The median time of first CR duration was 12 months (range, 6 to 40). All patients were reinduced with all-trans retinoic acid (ATRA), followed in 12 of 15 cases by mitoxantrone and Ara-C as consolidation. Fourteen patients received the BAVC (BCNU, Ara-C, m-AMSA, and VP-16) schedule as conditioning regimen. Unpurged marrows were collected immediately before conditioning treatment, analyzed by RT-PCR, and reinfused at median of 2 months (range, 2 to 7) from the achievement of second CR. Seven patients were PCR+ and eight PCR− for PML/RARα in their pretransplant marrows. All seven patients of the former group remained PCR+ during the follow-up and relapsed at a median time of 5 months (range, 2 to 9) from ABMT and 9 months (range, 4 to 14) from second CR. Of the eight PCR− patients, all remained PCR− during the follow-up controls. One patient relapsed at 10 months from ABMT, one died of a secondary (PML/RARα−) leukemia, and six are in hematologic and molecular remission at a median time of 28 months (range, 15 to 60) after ABMT and 32 months (range, 17 to 62) from second CR. Our results indicate that, in APL patients in second CR, ABMT with PML/RARα− marrow cells is likely to result in prolonged clinical and molecular remissions. Conversely, patients who test PCR+ after reinduction necessitate the use of alternative aggressive approaches, including unrelated allogeneic transplant.

scholarly journals Evidence of hepatitis in patients receiving transfusions of blood components containing antibody to hepatitis C

Blood ◽  
1993 ◽  
Vol 82 (3) ◽  
pp. 1000-1005 ◽  
Author(s):  
SK Aoki ◽  
PV Holland ◽  
LP Fernando ◽  
IK Kuramoto ◽  
S Anderson ◽  
...  
Keyword(s):  
Hepatitis C ◽  
Hcv Infection ◽  
Hcv Rna ◽  
Blood Components ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Immunoblot Assay ◽  
Polymerase Chain ◽  
Hepatitis C Virus Antibody

Abstract When hepatitis C virus antibody (anti-HCV) enzyme immunoassay (EIA1) testing became available in 1990, we tested samples from previously transfused blood units, traced the recipients of reactive units, and evaluated the recipients for HCV infection during the 12 months after transfusion. Ten of 42 recipients of EIA1-reactive blood were anti-HCV reactive on follow-up by EIA1 and 12 were reactive by a second- generation assay (EIA2). Reverse transcriptase-polymerase chain reaction (RT-PCR) detected HCV RNA in 5 seronegative recipients. In all, 17 of 42 recipients (40%) of EIA1-reactive blood had evidence of HCV infection. In comparison, 54 surgery patients, who received either no transfusion or autologous EIA1-nonreactive blood, remained EIA1 nonreactive and RT-PCR negative for 1 year; 1 patient (1.8%) became EIA2 reactive (P < or = .01). Of the recipients of anti-HVC reactive blood transfusions (reactive by both EIA1 and a supplemental 4-antigen strip immunoblot assay [RIBA2]), 14 (93%) of the recipients had evidence of HCV infection compared with only 3 of 27 recipients (11%) of EIA1-reactive, RIBA2-nonreactive blood (P < or = .01). Thus, blood components reactive for anti-HCV EIA1 may have transmitted HCV up to 40% of the time, but blood components found reactive by both EIA1 and RIBA2 may transmit HCV with a frequency of greater than 90%.

scholarly journals Improved RNA Extraction from Woody Plants for the Detection of Viral Pathogens by Reverse Transcription-Polymerase Chain Reaction

Plant Disease ◽  
1997 ◽  
Vol 81 (2) ◽  
pp. 222-226 ◽  
Author(s):  
Donald J. MacKenzie ◽  
Morven A. McLean ◽  
Srima Mukerji ◽  
Margaret Green
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Woody Plants ◽  
Rna Extraction ◽  
Viral Rna ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Apple Stem ◽  
Spin Column ◽  
Polymerase Chain

An efficient procedure for the extraction of high-quality RNA from woody plants without the use of phenol, organic solvents, or alcohol precipitation is described. The method employs commercially available spin-column matrices and mitigates the inhibitory effects of plant polysaccharides and polyphenolic compounds commonly observed on subsequent polymerase chain reaction amplification when conventional extraction methods are applied to woody plant species. The method described has been successfully used in the development of highly sensitive reverse transcription-polymerase chain reaction (RT-PCR) techniques for the detection of a number of viruses in their woody hosts. The viruses detected included apple stem grooving capillovirus (ASGV), apple stem pitting virus, Prunus necrotic ringspot ilarvirus (PNRSV), grapevine fanleaf and Arabis mosaic nepoviruses, and grapevine leafroll-associated closterovirus type 3. The method described was equally effective for the extraction of viral RNA from either budwood, leaves, or flower blossoms as determined by the equivalent RT-PCR detection of ASGV and PNRSV from these tissues. Detection of viral RNA in samples of total plant RNA prepared using this method was found to be as sensitive as was previously described for the immunocapture RT-PCR technique.

scholarly journals Evidence of hepatitis in patients receiving transfusions of blood components containing antibody to hepatitis C

Blood ◽  
1993 ◽  
Vol 82 (3) ◽  
pp. 1000-1005
Author(s):  
SK Aoki ◽  
PV Holland ◽  
LP Fernando ◽  
IK Kuramoto ◽  
S Anderson ◽  
...  
Keyword(s):  
Hepatitis C ◽  
Hcv Infection ◽  
Hcv Rna ◽  
Blood Components ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Immunoblot Assay ◽  
Polymerase Chain ◽  
Hepatitis C Virus Antibody

When hepatitis C virus antibody (anti-HCV) enzyme immunoassay (EIA1) testing became available in 1990, we tested samples from previously transfused blood units, traced the recipients of reactive units, and evaluated the recipients for HCV infection during the 12 months after transfusion. Ten of 42 recipients of EIA1-reactive blood were anti-HCV reactive on follow-up by EIA1 and 12 were reactive by a second- generation assay (EIA2). Reverse transcriptase-polymerase chain reaction (RT-PCR) detected HCV RNA in 5 seronegative recipients. In all, 17 of 42 recipients (40%) of EIA1-reactive blood had evidence of HCV infection. In comparison, 54 surgery patients, who received either no transfusion or autologous EIA1-nonreactive blood, remained EIA1 nonreactive and RT-PCR negative for 1 year; 1 patient (1.8%) became EIA2 reactive (P < or = .01). Of the recipients of anti-HVC reactive blood transfusions (reactive by both EIA1 and a supplemental 4-antigen strip immunoblot assay [RIBA2]), 14 (93%) of the recipients had evidence of HCV infection compared with only 3 of 27 recipients (11%) of EIA1-reactive, RIBA2-nonreactive blood (P < or = .01). Thus, blood components reactive for anti-HCV EIA1 may have transmitted HCV up to 40% of the time, but blood components found reactive by both EIA1 and RIBA2 may transmit HCV with a frequency of greater than 90%.

scholarly journals Genetic defects underlying paroxysmal nocturnal hemoglobinuria that arises out of aplastic anemia

Blood ◽  
1995 ◽  
Vol 86 (12) ◽  
pp. 4656-4661 ◽  
Author(s):  
S Nagarajan ◽  
RA Brodsky ◽  
NS Young ◽  
ME Medof
Keyword(s):  
Aplastic Anemia ◽  
Rna Splicing ◽  
Antithymocyte Globulin ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Rt Pcr ◽  
Chain Reaction ◽  
The Third ◽  
Mrna Transcripts ◽  
Polymerase Chain

Treatment of severe aplastic anemia with antithymocyte globulin (ATG) and cyclosporin leads to clinical remission in a large proportion of patients. As many as 10% to 57% of these patients, however, develop paroxysmal nocturnal hemoglobinuria (PNH). We and others have observed that this secondary PNH appears to be more indolent than classical PNH, which results from an acquired mutation in the PIG-A gene. In the present study, we compared PIG-A mRNA transcripts in affected cells from patients with secondary PNH and patients with classical PNH. All four of our aplastic patients who developed PNH had a negative Ham test at diagnosis. Two of the four showed a positive Ham test within 3 months after ATG/cyclosporin administration, one developed a positive test at 6 months, and another at 18 months after immunosuppressive therapy. All four patients remain transfusion-independent with no thrombotic episodes after a mean follow-up of 30 months (range, 6 to 63 months). Reverse transcription-polymerase chain reaction (RT-PCR) of PIG-A transcripts in DAF-/CD59- neutrophils or lymphocyte lines of the four patients showed PIG-A abnormalities in all cases. Transition of C163 to T was found in one, a 14-bp deletion (positions 1141 to 1154) was found in the second, deletion of C39 was found in the third, and two mutations, transition of C55 to T and transversion of T762 to A, were found in the fourth. These abnormalities compared with findings of abnormal RNA splicing causing a 133-bp deletion, a 4-bp insertion (between positions 578 and 579), loss of A767, and loss of C575 in four patients with primary PNH. We conclude that secondary PNH that evolves out of aplastic anemia, like classical PNH, is associated with mutations in the PIG-A gene. The apparent indolent nature of this disease probably reflects early detection.

scholarly journals Endothelin-1–Mediated Alteration of Metallothionein and Trace Metals in the Liver and Kidneys of Chronically Diabetic Rats

2002 ◽  
Vol 3 (3) ◽  
pp. 193-198 ◽  
Author(s):  
Lu Cai ◽  
Shali Chen ◽  
Terry Evans ◽  
M. George Cherian ◽  
Subrata Chakrabarti
Keyword(s):  
Trace Metals ◽  
Diabetic Rats ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Endothelin 1 ◽  
Liver And Kidney ◽  
Polymerase Chain ◽  
Age And Sex

In the present study, the role of endothelin-1 (ET-1) on alterations of hepatic and renal metallothionein (MT) and trace metals (Zn, Cu, and Fe) were investigated in streptozotocin (STZ)- induced diabetic rats. Diabetic rats, age- and sex-matched controls, as well as control and diabetic animals on a dualETA/ETBreceptor blocker, bosentan, were investigated after 6 months of follow-up. MT was measured by cadmium-heme assay. Metals were measured by atomic absorption spectrometer. ET-1 mRNA was analyzed by reverse transcriptase–polymerase chain reaction (RT-PCR) technique. Hepatic and renal ET-1 mRNA was increased in diabetic rats as compared to control rats, along with an increase in both hepatic and renal MT proteins. The increased hepatic MT protein level was associated with decreases in hepatic Cu and Fe, whereas increased renal MT was associated with increases in renal Cu and Fe accumulation. Zn levels were unaltered in both organs in diabetic rats. Bosentan treatment partially prevented the increase in MT levels in both liver and kidney, along with reduced serum creatinine and increased urinary creatinine levels. Further bosentan treatment corrected the increased Cu and Fe levels in the kidney in diabetic rats, but reduced hepatic Cu and Fe levels. No significant effects of bosentan treatment on nondiabetic rats were observed. The data suggest that the possible effects of ET antagonism in diabetes may be mediated via changes in MT and trace metals.

Early Detection of Relapse by Prospective Reverse Transcriptase-Polymerase Chain Reaction Analysis of the PML/RARα Fusion Gene in Patients With Acute Promyelocytic Leukemia Enrolled in the GIMEMA-AIEOP Multicenter “AIDA” Trial

Blood ◽  
1998 ◽  
Vol 92 (3) ◽  
pp. 784-789 ◽  
Author(s):  
Daniela Diverio ◽  
Vincenzo Rossi ◽  
Giuseppe Avvisati ◽  
Silvia DeSantis ◽  
Alessandra Pistilli ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Fusion Gene ◽  
Promyelocytic Leukemia ◽  
Rt Pcr ◽  
Molecular Monitoring ◽  
Chain Reaction ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Polymerase Chain

Abstract Although the majority of patients with acute promyelocytic leukemia (APL) are potentially cured by treatments combining all-trans retinoic acid (ATRA) and chemotherapy (CHT), a sizable proportion (around 30%) will relapse during follow-up. Retrospective molecular monitoring studies using reverse transcriptase-polymerase chain reaction (RT-PCR) for the specific PML/RARα fusion gene, have shown that a positive test usually precedes the occurrence of hematologic relapse. Prospective RT-PCR analyses were performed since 1993 at diagnosis and at preestablished time intervals during follow-up in bone marrow (BM) samples of 163 patients with PML/RARα+ APL enrolled in the multicenter Gruppo Italiano Malattie Ematologiche Maligne dell' Adulto (GIMEMA) trial AIDA (All-trans retinoic acid plus Idarubicin). Treatment consisted of ATRA and idarubicin for induction followed by three polychemotherapy courses as consolidation. The sensitivity level of the RT-PCR assay for PML/RARα, as assessed by serial dilution experiments, was 10−4. All patients were in hematologic remission and tested PCR− at the end of consolidation. Of 21 who converted to PCR-positive thereafter, 20 underwent hematologic relapse at a median time of 3 months (range, 1 to 14) from the first PCR+ result. Seventeen of these 21 (81%) PCR+ conversions were recorded within the first 6 months postconsolidation. Of 142 who tested persistently PCR− in ≥2 tests after consolidation, 8 had hematologic relapse and 134 remained in complete remission (CR) after a median follow-up of 18 months (range, 6 to 38) postconsolidation. Using a time-dependent Cox model, the relative risk of hematologic relapse of patients who converted to PCR+ was 31.8 (confidence limits 95%, 12.9 to 78.3). Our results indicate that conversion to PCR positivity for PML/RARα during remission is highly predictive of subsequent hematologic relapse and highlight the prognostic value of stringent molecular monitoring during the early postconsolidation phase in APL. As a result of the present study, salvage treatment in patients enrolled in the GIMEMA trial AIDA is now anticipated at the time of molecular relapse, defined as the conversion to PCR positivity in two successive BM samplings during follow-up. © 1998 by The American Society of Hematology.

scholarly journals Characteristics of recovered COVID-19 patients with recurrent positive RT-PCR findings in Wuhan, China: a retrospective study

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Tie-Jun Shui ◽  
Chao Li ◽  
Hong-bing Liu ◽  
Xiaohua Chen ◽  
Bi-ke Zhang
Keyword(s):  
Polymerase Chain Reaction ◽  
Retrospective Study ◽  
Clinical Characteristics ◽  
Throat Swab ◽  
Antibody Detection ◽  
Controlled Trials ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

Abstract Background Two months after the outbreak of coronavirus disease 2019 (COVID-19) in Wuhan, China, tens of thousands of hospitalized patients had recovered, and little is known about the follow-up of the recovered patients. Methods The clinical characteristics, reverse transcriptase-polymerase chain reaction (RT-PCR) results from throat swab specimens and the results of serological COVID-19 rapid diagnostic test (RDT) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were retrospectively reviewed for a total of 758 recovered patients who were previously hospitalized in 17 hospitals and quarantined at 32 rehabilitation stations in Wuhan, China. Results In total, 59 patients (7.78%) had recurrent positive findings for COVID-19 on RT-PCR from throat swabs. With regard to antibody detection, 50/59 (84.75%) and 4/59 (6.78%) patients had positive IgG or dual positive IgG/IgM RDT results, respectively. Conclusions Some patients who had been quarantined and had subsequently recovered from COVID-19 had recurrent positive RT-PCR results for SARS-CoV-2, and the possibility of transmission of the virus by recovered patients needs further investigation. Trial registration Current Controlled Trials ChiCTR2000033580, Jun 6th 2020. Retrospectively registered.

Sign in / Sign up

Export Citation Format

Share Document