RNA extracts with polyadenylic acid sequences transfer specific sensitivity for a low molecular weight antigen (MW 486).
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Systematic comparison of quantity and quality of RNA recovered with commercial FFPE tissue extraction kits.
BACKGROUND: FFPE tissue samples are commonly used in biomedical research and are a valuable source for next-generation sequencing in oncology, however, extracting RNA from these samples can be difficult the quantity and quality achieved can impact the downstream analysis. This study compared the effectiveness of seven different commercially available RNA extraction kits specifically designed for use with FFPE samples in terms of the quantity and quality of RNA recovered. METHODS: This study used 9 samples of FFPE tissue from three different types of tissue (Tonsil, Appendix and lymph node of B-cell lymphoma) to evaluate RNA extraction methods. Three sections of 20 µm of each sample were combined per sample. The slices were distributed in a systematic manner to prevent any biases. Each of the 7 commercially available RNA extraction kits were used according to manufacturer's instructions, with each sample being tested in triplicate resulting in a total of 189 extractions. The concentration, RNA quality score (RQS) and DV200 of each extraction was analysed using a nucleic acid analyser to determine the quantity and quality of the recovered RNA. RESULTS: This study found that despite processing the FFPE samples in the same standardized way, there were disparities in the quantity and quality of RNA recovered across the different tissue types. Additionally, the study found notable differences in the quantity of RNA recovered when using different extraction kits. In terms of quality, three of the kits performed better than the other four in terms of RQS and DV200 values. CONCLUSION: Though many laboratories have developed their own protocols for specific tissue types, using commercially available kits is still a popular option. Although these kits use similar processes and extraction procedures, the amount and quality of RNA obtained can vary greatly between kits. In this study, among the kits tested, while the Roche kit, provided a nearly systematic better-quality recovery than other kits, the ReliaPrep FFPE Total RNA miniprep from Promega yielded the best ratio of both quantity and quality on the tested tissue samples.
Paraffin Embedding↗
Translation of silk fibroin messenger RNA in an Ehrlich ascites cell-free extract.
RNA identified by its base composition and T1 RNase oligonucleotide pattern as the message for silk fibroin was purified from mature posterior silk glands of Bombyx mori larvae and used to direct polypeptide synthesis in an Ehrlich ascites cell-free extract. Fibroin mRNA stimulated [3-H]alanine incorporation about 3- to 4-fold in the presence of 80 mM K+ and 4 mM Mg-2+. The stimulation was reduced in the presence of 5 times 10-minus 6 to 10-minus 4 M aurintricarboxylic acid, an inhibitor of the initiation of protein synthesis. The cell-free products were heterogeneous in size, including peptides as large as 100,000 daltons. They co-precipitated with carrier fibroin sequences after digestion with trypsin. A large fraction of the polypeptides synthesized in response to fibroin mRNA was precipitated by antiserum directed against amino acid sequences in noncrystalline region polypeptides of fibroin. Furthermore, after digestion with chymotrypsin, a major fraction of the cell-free products specifically co-precipitated with crystalline region sequences of native fibroin. The size and amino acid composition of the fibroin crystalline region polypeptides isolated from the cell-free products were similar to those from native fibroin.
Amino Acids↗
[Biochemical and cytospectrophotometric analysis of the extraction of RNA from spinal cord cell structures].
By means of two-wavelength spectrophotometry, according to Tsanev and Markov, a stability of RNA content has been demonstrated in rabbit spinal cord sections treated with cold perchloric acid: it was only after 18 and particularly 48 hr incubation of the section in a 16% perchloric acid solution that the total tissue RNA began to be extracted. Cytospectrophotometrical study of the motoneurons of spinal cord anterior horns and perineuronal glial cells in gallocyanin -- chrome alum stained sections has shown a rapid loss of RNA under effect of the cold perchloric acid: as early as after a 2 hr treatment, about 2/3 of the whole cellular RNA was extracted from the motoneurons, while about 1/2 from their glial satellite cells. Hydrolysis of the rest of RNA was found out in the neurons and in the neuroglia only after a 18 hr extraction with the perchloric acid. Similarities and differences in the features of neuronal and glial RNA are discussed.
Animals↗
Poly(A)+ RNA from Tetrahymena: stimulation of protein synthesis in vitro.
Cell-free synthesis of high molecular weight polypeptides, programmed by RNA from Tetrahymena pyriformis strain W is reported, and methods for preparation of the RNA are described. The RNA was extracted by the SDS-phenol-chloroform-isoamyl alcohol technic. The bulk of extracted RNA was ribosomal and on sucrose gradients peaked at approximately 17S and 25S. After heat denaturation all the 25S RNA was converted to 17S, indicating the presence of hidden breaks, possibly the result of nuclease activity during extraction. Nevertheless, when poly(A) +/- RNA was collected using oligo-(dT)-cellulose column chromatography, it promoted a 15-fold increase in incorporation of [35S] methionine into TCA-precipitable material. Slab-gel electrophoresis and autoradiography of the product revealed 12 different major polypeptides, varying in weight from 28,000 to 65,000 Daltons. A method for preparation of translatable RNA from Tetrahymena will make possible the comparison of messenger RNAs associated with specific cell structures and with different developmental events.
Animals↗
Surface immunoglobulins of lymphocytes in mouse plasmacytoma. IV. Evidence for the persistence of the effect of plasmacytoma-RNA on the surface immunoglobulins of normal lymphocytes in vivo and in vitro.
The previous findings were confirmed that RNA extracted from murine plasmacytoma alters the character of the lymphocyte surface immunoglobulins (SIg) to express the idiotypic specificity of the Ig of the plasmacytoma from which the RNA was derived (cell conversion). RNA extracted from spleens of plasmacytoma-RNA-injected BALB/c mice also had convering activity, and if injected into other mice, caused the appearance of RNA active in cell conversion in spleens of the second set of mice. This activity was lost only after two additional transfers. When splenic cells from animals 1 hr after injection with RNA extracted from MOPC 300, LPC-1 or MOPC 104E, were cultured for 7 days, the proportion of cells with the SIg specific for these tumours increased. The cell-converting activity of the RNA extracted from the cultured cells after 7 days incubation ('7-day' RNA) was higher than that of RNA extracted from cells after 1 hr incubation ('immediate' RNA). 'Seven-day' RNA could be used for sequential transfers without marked loss of activity in cell conversion for at least five transfers. The repetitive transferability of this phenomenon by the injection of plasmacytoma-RNA suggests the possibility of RNA replication in the recipient cells.
Animals↗
Transfer of experimental allergic orchitis with immune RNA studies in vivo.
Ribonucleic acid extracts (RNA) obtained from the lymph nodes and spleens of guinea pigs, which were immunized with testicular antigen emulsified in Freund's complete adjuvant (FCA), were injected intraperitoneally into normal guinea pigs. The transferred guinea pigs developed a delayed hypersensitivity to sperm antigens and testicular lesions which resembled the lesions obtained in the donor RNA guinea pigs. When the transfer was performed with RNA extracted from guinea pigs immunized with FCA alone or with 'immune' RNA treated with Ribonuclease, neither cellular immunity nor testicular lesions were observed.
Animals↗
Stimulation of 3H-uridine incorporation into ribosomal ribonucleic acids by insulin.
Ribosomal RNA was extracted from hepatic ribosomal subunits of rats treated with alloxan or with alloxan and insulin and additionally injected with 3H-uridine. Sedimentation of the extracted RNA through sucrose, gradients with registration of the optical density and with measurement of the radioactivity of rRNA reveals reduction of uridine incorporation after the induction of diabetes of diabetes mellitus. If alloxan-diabetic animals are substituted with insulin, this decrease in uridine incorporation is reversed to incorporation rates which even exceed uridine incorporation of control animals. With regard to the reduction of protein synthesis in diabetes mellitus this finding of reduced rRNA synthesis is suggested to be an additional factor in the reduction of ribosomal aggregation.
Animals↗
[Influence of temperature on the preferential extraction of RNA polymerase I from hepatic nuclei of the rat].
RNA polymerase I has been extracted from rat liver nuclei by three consecutive washings at 0 degrees C with a medium of relatively low ionic strength (0.15 M KCl) containing Mg++ rather than by incubating the organelles at 37 degrees C in the same medium, as originally proposed by Chesterton and Butterworth. The modified technique, which has the advantage of preventing a temperature-mediated conversion of form IB to IA, gives similar yields of RNA polymerase I and retains the capacity of preferentially extracting the enzyme with respect to the other forms of nuclear RNA polymerase.
Animals↗
A new rapid method for selective extraction of RNA from fixed mammalian tissues.
Treatment of formalin-fixed mammalian tissues with concentrated or 50% phosphoric acid at 5 degrees C for 20 and 50 min. respectively reveals complete extraction of RNA as judged by methyl green followed by staining with pyronin. This procedure also causes depolymerisation of DNA as indicated by the red staining of the nuclei. Sections treated with concentrated phosphoric acid at 5 degrees C for 30 min. causes disruption of the double helical structure of DNA what results in the depression of the pyronin staining. Similarly treated sections show Feulgen positive nuclei. Treatment of sections in 25 % phosphoric acid at 60 degrees C for 15 min. followed by staining with methyl green and pyronin show red nuclei, nucleoli and the cytoplasm. This indicates that extraction of RNA is only possible in cold and not at elevated temperature.
Animals↗
Processing of RNA by cytoplasmic extract. Synthesis and processing of RNA by isolated HeLa cell nuclei incubated with cytoplasmic extract.
Nuclei are isolated from HeLa cells by a low speed centrifugation procedure. These nuclei can carry out RNA synthesis at 30 degrees. The effect of cytoplasmic extract on the transport and processing of RNA is measured. In the presence of cytoplasmic extract the precursor to 4S RNA is processed to 4S RNA and an RNA species OF 7-9S is transported outside the nuclei.
Cell Extracts↗
Mediation of immune responses to tumor antigens in vitro by immune RNA.
It was shown that normal nonimmune C3H mouse spleen cells became specifically cytotoxic to chemically-induced syngeneic C3H tumor cells by incubation with xenogeneic I-RNA extracted from the lymphoid organs of specifically immunized guinea pigs. This response was specific for the tumor used to immunize the I-RNA donor. In a totally syngeneic system, we showed that syngeneic I-RNA extracted from the spleens of tumor-bearing rats mediated cytotoxic immune reactions which were directed specifically against the tumor-associated antigens of syngeneic rat tumor target cells. Active antitumor I-RNA synthesis in the lymphoid organs of I-RNA donor animals reached a maximum between days 14 and 21, depending on the route of administration and the nature of the immunizing tumor. Active I-RNA preparations were insensitive to treatment with deoxyribonuclease or pronase, but were inactivated by ribonuclease treatment; thereby indicating that the active moiety was one or more species of RNA. The active fractions of the I-RNA preparations had sedimentation values in sucrose density gradients of 12-16S, and comprised only a small fraction of the total RNA present in the lymphoid cells. Active antitumor I-RNA appeared to be localized in the cytoplasm of sensitized lymphoid cells, rather than in the nucleus. Lymphocytes from normal human donors as well as from cancer patients, when incubated with xenogeneic or allogeneic I-RNA, became specifically cytotoxic for human tumor cells in vitro. Crossreactivity among tumors of the same histologic type was observed, but not crossreactivity with tumors of other histologic types. Xenogeneic I-RNA extracted from the lymphoid organs of donor animals immunized either iwth tumor cells or normal tissues, following incubation with normal allogeneic lymphocytes, mediated cytotoxic immune reactions which were directed both against tumor-associated antigens and normal transplantation antigens. However, when autologous lymphocytes were used as effector cells, only immune reactions directed against tumor-associated antigens were observed. Allogeneic I-RNA extracted from peripheral blood lymphocytes of human cancer patients mediated specific cytotoxic immune reactions that were directed against common tumor-associated antigens shared by human tumors of similar histologic type. I-RNA's directed against "self" normal cell surface antigens appear to be recognized as self by lymphocytes, and immune responses against these self antigens are not elicited. On the other hand, I-RNA's directed against "nonself" tumor-associated antigens induce lymphocytes to effect specific antitumor immune responses. Our data are consistent with the hypothesis that I-RNA is an information-containing ribonucleic acid molecule capable of mediating immune reactions in vitro which are specific for the tumor-associated antigens of the tumor used to immunize the I-RNA donor.
Animals↗
Transcription of the Marek's disease virus genome in virus-induced tumors.
Transcription of the Marek's disease virus (MDV) genome in tumor tissues from MDV-infected chickens has been studied by analyzing the hybridization kinetics of (3)H-labeled MDV DNA with unlabeled RNA extracted from these tissues. Lymphoid tumors of ovary, spleen, liver, and kidney contained MDV genomes, but the virus-specific RNA sequences were transcribed from less than 15% of the viral DNA. A virus nonproductive lymphoblastoid cell line, designated MKT-1, has been established from a kidney lymphoma and contains 15 MDV genomes per cell. In these cells, 12 to 14% of the viral DNA was transcribed. Thus transcription of the MDV genome was restricted both in tumor tissues and MKT-1 cells. A hybridization experiment where RNA extracted from MKT-1 cells and RNA extracted from a spleen tumor were mixed and hybridized to (3)H-labeled MDV DNA indicated that the virus-specific RNAs from the two sources were encoded by the same DNA sequences. The polyribosomal fractions of MKT-1 cells and this spleen tumor contained only a portion of the virus-specific RNA sequences found in whole-cell extracts, indicating the existence of a posttranscriptional control mechanism which prevents the transfer of certain viral RNA transcripts to the polyribosomes. The data suggest that the repressed expression of the viral genome in lymphoid tumor tissues and MKT-1 cells may be the result of precise controls within the cell at the transcriptional and posttranscriptional levels.
Animals↗
Effect of RNase III on efficiency of translation of bacteriophage T7 lysozyme mRNA.
RNase III had no positive effect on the translation of bacteriophage T7 lysozyme mRNA in vivo or in vitro. The time of appearance and quanity of lysozyme in T7-infected E. coli BL107, an RNase III- strain, and T7-infected E. coli BL15, a nearly isogenic RNase III+ strain, were indistinguishable. Nearly identical patterns of lysozyme mRNA activity were obtained when RNA extracted at different times after infection of RNase III+ and RNase III- hosts was translated in cell-free extracts of E. coli containing or lacking RNase III. Exposure of RNA extracted from T7-infected E. coli BL107 (RNase III-) to purified RNase III did not increase the lysozyme mRNA activity of this RNA. The only result that implied that RNase III has a differential effect on the translatability of the lysozyme mRNA was the translation of fractionaed RNA from T7-infected E. coli BL107. Translation of the smallest and largest lysozyme messages, 0.33 x 10(6) and 4 x 10(6) to 5 x 10(6) daltons, was the most inefficient in RNase III- cell-free extracts as compared to RNase III+ cell-free translation. The translation of the most abundant, medium-sized lysozyme mRNA between 0.9 x 10(6) and 1.5 x 10(6) daltons was the least affected by the absence of RNase III. The existence of a lag between the appearance of lysozyme mRNA and the appearance of lysozyme in T7 infection was confirmed. In these studies a very rapid method of RNA extraction was used, eliminating the possibility of continued RNA transcription during cell collection and RNA extraction. With this method of analysis, the length of the lag period was established at about 3 min. The possibility that RNase III is the controlling element of the lag period was eliminated by these investigations.
Cell-Free System↗
Mediation of cytotoxic immune responses against human tumor-associated antigens by xenogeneic immune RNA.
Xenogeneic immune RNA (I-RNA), extracted from the lymphoid organs of sheep or guinea pigs immunized with human tumor cells, mediated in vitro cytotoxic immune responses that were directed specifically against tumor-associated antigens of human tumor target cells. Normal human peripheral blood lymphocytes from healthy donors became markedly more cytotoxic for human tumor target cells after being incubated with I-RNA extracted from the lymphoid organs of animals that had been immunized with that particular tumor. Gastric carcinoma, malignant melanoma, and carcinoma of the breast were studied. Lymphocytes incubated with RNA from animals immunized with only complete Freund's adjuvant evidenced no increased cytotoxic activity. RNA extracted from the lymphoid organs of animals immunized with normal skin fibroblasts that were autologous to the immunizing tumor, when incubated with normal allogeneic lymphocytes, also mediated cytotoxic immune reactions against tumor target cells. These immune responses probably were directed principally against normal transplantation antigens. However, when lymphocytes that were autologous to the immunizing tumor and/or the tumor target cells were incubated with RNA from animals immunized with autologous normal fibroblasts, cytotoxicity did not increase. Only I-RNA extracted from donor animals specifically immunized with tumor cells mediated cytotoxic antitumor immune responses when incubated with autologous lymphocytes.
Animals↗
Evidence that the regulation of diphtheria toxin production is directed at the level of transcription.
It has been known for several decades that iron inhibits the production of diphtheria toxin by Corynebacterium diphtheriae by preventing expression at maximal levels. We examined the inhibition kinetics of toxin production after the addition of either iron or rifampin to iron-limited cultures of C7 (betatox+). Iron-mediated inhibition of toxin production was found to be linear within the range of 16 nM to 16 micron. The inhibition kinetics following the addition of iron or rifampin was almost identical. [3H]RNA extracted from iron-limited toxigenic C. diphtheriae was found to hybridize to a greater extent to corynephage beta DNA than either [3H]RNA extracted from toxigenic C. diphtheriae before the onset of toxin production or [3H]RNA extracted from nonlysogenic, nontoxigenic C. diphtheriae.
Corynebacterium diphtheriae↗
Genome expression and mRNA maturation at late stages of productive adenovirus type 2 infection.
RNA from adenovirus 2-infected KB cells was annealed in liquid with RNA in vast excess to viral heavy (l) and light (r) 32P-labeled DNA strands. Hybridization kinetics were analyzed by computer to estimate the number of viral RNA abundance classes, their relative concentrations, and the fraction of each DNA strand from which they originated. Early whole cell RNA extracted 5 h postinfection annealed rapidly to 10 to 15% of l and r strands and then slowly to final values of 60 and 40% of l and r strands. By 9 h postinfection the expression of late genes was apparent and whole cell RNA annealed to 20 and 75% of l and r strands. Whole cell RNA extracted between 12 and 36 h postinfection annealed to 7 to 15% and 75 to 90% of l and r strands. Late nuclear RNA hybridized to 10 and 90% of l and r strands, and late polyribosomal RNA hybridized to 20 and 75% of l and r strands. Based upon kinetic analyses, we estimate that mRNA synthesized exclusively during late stages arises from about 6 to 8% and 45 to 49% of l and r strands. This assumes that the early class I mRNA (in low concentration late) originates from 8 to 10% and 6 to 10% of l and r strands and that early class II mRNA (in high concentration late) is derived from 2% and 8 to 13% of l and r strands. Mixing experiments indicated that early mRNA is a subset of RNA extracted from polyribosomes late after infection and that late nuclear RNA contains sequences complementary to early l strand class I nRNA. RNA-RNA hybrids were isolated from late mRNA containing sequences from 60% of l and r strands, but it is not known when these were synthesized, and therefore whether complementary RNA transcripts are synthesized late after infection, as they are known to be synthesized early. These results demonstrate that portions of the genome are transcribed into RNA sequences that remain confined to the nucleus and are not exported to polyribosomes as mRNA.
Adenoviruses, Human↗
Pre-proparathyroid hormone: fidelity of the translation of parathyroid messenger RNA by extracts of wheat germ.
Pre-proparathyroid hormone is the major protein synthesized in wheat-germ extracts in response to addition of an 8-15S fraction of parathyroid RNA. The accuracy of the translation of the mRNA from parathyroid tissue was examined by analysis of the carboxyl-terminal tryptic peptide and the amino-terminal amino acid of the protein, by analysis of the size distribution of the mRNA, and by translation of the mRNA in a second cell-free extract. When 8-15S RNA was fractionated on a sucrose gradient containing formamide, RNA that supported the synthesis of pre-proparathyroid hormone was present in a single symmetrical peak, suggesting that it was homogeneous. Analyses by paper chromatography and electrophoresis of the proline-containing tryptic peptides of pre-proparathyroid hormone indicate that they are identical with the corresponding proline-containing peptides of parathyroid hormone. Because the COOH-terminal tryptic peptide of parathyroid hormone contains proline, the data indicate that the COOH termini of pre-proparathyroid hormone and parathyroid hormone are identical. Methionine from initiator [35S]Met-tRNAfMet was rapidly incorporated into pre-proparathyroid hormone by the wheat-germ extract, and a single-step Edman degradation selectively removed almost all of the initiator [35S]methionine present in pre-proparathyroid hormone. Translation of the 8-15S RNA in a cell-free extract from Krebs-II ascites cells resulted in a protein that comigrated with pre-proparathyroid hormone on sodium dodecyl sulfate-acrylamide gel electrophoresis. These data support the conclusion that the wheat-germ system accurately translates the mRNA for parathyroid hormone, and they strengthen the contention that pre-proparathyroid hormone is the initial biosynthetic product.
Animals↗