Total RNA (600 ng) was ligated to 5 μM of 5′-adenylated, 3′-blocked adaptor (Universal miRNA Cloning Linker, New England BioLabs) with 280 units of T4 RNA ligase, Truncated KQ (New England BioLabs), 25% PEG 8000 and 1 μl of RNaseOUT (Life Technologies) in a 20-μl reaction at 25 °C for 16–24 h. After cleanup with RNA Clean and Concentrator columns (Zymo Research), followed by DNase treatment, cDNA was synthesized with 5 pmol of universal RT primer (Supplementary Table 6 ) and SuperScript III reverse transcriptase. PCR amplification was carried out using 5 μM of the TERC_L2 and universal RT or TERC_L3 and universal RT primer sets (Supplementary Table 6 ) with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad). PCR products were directly analyzed on 2.5% agarose gels to visualize mature TERC and extended TERC transcripts or subjected to QIAquick PCR purification columns (Qiagen) for library preparation for deep sequencing. For Sanger sequencing, 3′ RACE PCR products were directly cloned into the pCR4_TOPO vector (Life Technologies), and individual clones were sequenced using the TERC_L2 or TERC_L3 primer.
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Telomerase RNA component
Telomerase RNA component
Telomerase RNA component is a non-coding RNA molecule that serves as the template for the addition of telomeric repeats to the ends of chromosomes.
It is a crucial component of the telomerase enzyme complex, which plays a key role in maintaining telomere length and cellular immortality.
This RNA component provides the template for the reverse transcription of telomeric DNA sequences, ensuring the preservation of telomeres during cell division.
Understanding the structure, function, and regulation of the Telomerase RNA component is essential for research into cellular aging, cancer, and stem cell biology.
It is a crucial component of the telomerase enzyme complex, which plays a key role in maintaining telomere length and cellular immortality.
This RNA component provides the template for the reverse transcription of telomeric DNA sequences, ensuring the preservation of telomeres during cell division.
Understanding the structure, function, and regulation of the Telomerase RNA component is essential for research into cellular aging, cancer, and stem cell biology.
Most cited protocols related to «Telomerase RNA component»
Clone Cells
Cloning Vectors
Deoxyribonucleases
DNA, Complementary
DNA Library
Gels
MicroRNAs
Oligonucleotide Primers
polyethylene glycol 8000
RNA-Directed DNA Polymerase
RNA Ligase (ATP)
Sepharose
SYBR Green I
telomerase RNA component
trioctyl phosphine oxide
Biological Assay
Cells
Chemiluminescence
GAPDH protein, human
Genome
Keratinocyte
Oligonucleotide Primers
prisma
Reverse Transcriptase Polymerase Chain Reaction
Sepharose
Skin
SYBR Green I
Telomerase
telomerase RNA component
Telomere
TERT protein, human
Tissue, Membrane
trizol
Individual eukaryotic cell-free protein synthesis systems differ in their composition and reaction conditions. Sf21- and CHO-based cell-free reactions have similar characteristics and were performed in a batch-formatted reaction mode. A 25 µL standard translation reaction of a Sf21 and CHO based cell-free synthesis was composed of 6 µL purified mRNA, 40% lysate, canonical amino acids (200 µM each), ATP (1.75 mM), GTP (0.45 mM) and 14C-labeled leucine (200 dpm/pmol) for the detection of de novo synthesized proteins. For the functional analysis of WNT proteins (β-catenin accumulation assay), proteins were synthesized in the absence of 14C-leucine. Protein translation reactions based on Sf21 lysates were incubated for 90 min at 27 °C, 600 rpm using a thermomixer (Eppendorf, Hamburg, Germany). Translation reactions based on CHO cell lysates were performed at 30 °C and 120 min with gentle shaking at 600 rpm. If required translation mixture (TM) of both cell-free reactions were further fractionated for analysis of protein translocation. The fractionation was realized by centrifugation at 16,000× g for 10 min at 4 °C in order to separate the ER-derived microsomal fraction (MF) of the cell lysate from the supernatant (S). The microsomal fraction was resuspended in PBS buffer without calcium and magnesium ions for further analysis.
To decrease the phosphorylation of eukaryotic translation factor eIF2α and thereby improving the capacity of cap-dependent translation initiation, the influence of the small component C38 (GSK2606414, GlaxoSmithKline, Dresden, Germany), a specific PERK inhibitor, was analyzed. Inhibitor containing lysate was preincubated for 10 min at RT before the cell-free reaction was started. To analyze the effect on the protein synthesis rate, the pIX4.0-Luc plasmid was added to the reaction and the yield of active luciferase was determined.
Synthesis in wheat germ lysate was performed in a transcription/translation coupled dialysis system using the RTS100 Wheat Germ CECF Kit (Biotechrabbit) for 24 h at 24 °C according to the manufacturer´s instructions. Again, 14C-leucine (2.47 dpm/pmol) was added to the reaction mixture to determine the size, yield and integrity of the de novo synthesized protein.
To obtain functional active hTERT enzyme, the assembly with a typical RNA component of telomerase is required [27 (link)]. The assembly was realized in two different ways: RNA was generated in a previous transcription reaction by using T7 RNA–polymerase followed by RNA purification using DyeEx spin columns (Qiagen). The freshly synthesized hTR RNA was directly added to the cell-free reaction (CHO and Sf21 cell-free reactions). Alternatively, a plasmid harboring the nucleotide sequence encoding the RNA component of human telomerase under control of the T7-promoter was added directly to the translation reaction (coupled reaction; wheat germ; final concentration of hTR plasmid: 20 ng/µL).
To decrease the phosphorylation of eukaryotic translation factor eIF2α and thereby improving the capacity of cap-dependent translation initiation, the influence of the small component C38 (GSK2606414, GlaxoSmithKline, Dresden, Germany), a specific PERK inhibitor, was analyzed. Inhibitor containing lysate was preincubated for 10 min at RT before the cell-free reaction was started. To analyze the effect on the protein synthesis rate, the pIX4.0-Luc plasmid was added to the reaction and the yield of active luciferase was determined.
Synthesis in wheat germ lysate was performed in a transcription/translation coupled dialysis system using the RTS100 Wheat Germ CECF Kit (Biotechrabbit) for 24 h at 24 °C according to the manufacturer´s instructions. Again, 14C-leucine (2.47 dpm/pmol) was added to the reaction mixture to determine the size, yield and integrity of the de novo synthesized protein.
To obtain functional active hTERT enzyme, the assembly with a typical RNA component of telomerase is required [27 (link)]. The assembly was realized in two different ways: RNA was generated in a previous transcription reaction by using T7 RNA–polymerase followed by RNA purification using DyeEx spin columns (Qiagen). The freshly synthesized hTR RNA was directly added to the cell-free reaction (CHO and Sf21 cell-free reactions). Alternatively, a plasmid harboring the nucleotide sequence encoding the RNA component of human telomerase under control of the T7-promoter was added directly to the translation reaction (coupled reaction; wheat germ; final concentration of hTR plasmid: 20 ng/µL).
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Adult
Biopsy
Cells
Culture Media, Serum-Free
dispase
Donors
Edetic Acid
Epidermis
Epithelial Cells
Ethics Committees, Research
Feeder Cells
Fibroblasts
Gluteal Region
Homo sapiens
isolation
Keratinocyte
Skin
Tissues
Trypsin
Native DNA-PKcs and Ku70/80 were purified to homogeneity from HeLa cells as described previously (19 (link)). The EMSA binding reactions were carried out in 10 μl volume containing 100 mM NaCl, 10 mM Tris, pH 7.5, 5% (w/v) glycerol, 1 mM MgCl2, 0.8 U of RNAse OUT (Invitrogen) and 0.33 pmol of 32P-labeled hTR probe. The amount of purified proteins used in each EMSA reaction is indicated in the figure legends. Following a 10 min incubation at 25°C, the reactions were fractionated on 5% non-denaturing PAGE, in 0.5× TBE for 90 V for 3 h. For the smaller truncated hTRs, the gels were run for 1 h.
For the EMSA experiments with DNA, a double-stranded 36 bp blunt-ended oligonucleotide corresponding to the sequence of a non relevant gene (PIG3) was used as the radiolabeled probe. The DNA probe was end-labeled with 32P using T4 kinase as described previously (20 (link)), and approximately 0.2 pmol of labeled probe was used for the binding reactions. For the DNA gel shift, the gels were run for 1 h.
The EMSA gels were dried on Whatman 3MM paper and exposed to X-ray films. Addition of competitor RNA, DNA or antibodies to the EMSA reactions, either before or 5 min after the radiolabeled probe, did not alter the results. The monoclonal antibodies used included anti-Ku70 and anti-Ku80 (Ab5 and Ab2, NeoMarkers) and anti-Myc (Santa Cruz Biotechnology).
For the EMSA experiments with DNA, a double-stranded 36 bp blunt-ended oligonucleotide corresponding to the sequence of a non relevant gene (PIG3) was used as the radiolabeled probe. The DNA probe was end-labeled with 32P using T4 kinase as described previously (20 (link)), and approximately 0.2 pmol of labeled probe was used for the binding reactions. For the DNA gel shift, the gels were run for 1 h.
The EMSA gels were dried on Whatman 3MM paper and exposed to X-ray films. Addition of competitor RNA, DNA or antibodies to the EMSA reactions, either before or 5 min after the radiolabeled probe, did not alter the results. The monoclonal antibodies used included anti-Ku70 and anti-Ku80 (Ab5 and Ab2, NeoMarkers) and anti-Myc (Santa Cruz Biotechnology).
Antibodies
DNA Probes
Electrophoretic Mobility Shift Assay
Gels
Genes
Glycerin
HeLa Cells
Magnesium Chloride
Monoclonal Antibodies
Native Polyacrylamide Gel Electrophoresis
Oligonucleotides
Phosphotransferases
PRKDC protein, human
Proteins
ribonuclease U
Sodium Chloride
telomerase RNA component
Tromethamine
X-Ray Film
Xrcc6 protein, human
Most recents protocols related to «Telomerase RNA component»
All in vivo and in vitro experiments were carried out with the S. aureus strain USA300. S. aureus was cultivated in brain heart infusion (BHI) medium (Thermo Fisher Scientific, Waltham, MA, US) at 37°C while shaking at 150 rpm. The bacteria were harvested during the mid-logarithmic phase and adjusted to the appropriate optical density (OD) at 600nm for the respective experiments in phosphate-buffered saline (PBS). The bacteria were then either used freshly for in vivo infection of mice or stored at -80 °C until further usage in in vitro experiments.
For heat-killing, the adjusted bacteria stock was incubated at 95°C for 5 minutes while shaking at 500 rpm. A fraction of the heat-killed bacteria was plated on Müller-Hinton (MH) plates. Plates were incubated for 5 days at 37°C to confirm heat-killing of the bacteria.
For heat-killing, the adjusted bacteria stock was incubated at 95°C for 5 minutes while shaking at 500 rpm. A fraction of the heat-killed bacteria was plated on Müller-Hinton (MH) plates. Plates were incubated for 5 days at 37°C to confirm heat-killing of the bacteria.
Mouse in vivo experiments were approved by the Office for Consumer Protection of Thuringia (TV-Number: UKJ-19-028 and UKJ-22-023).
Third-generation (G3) female Terc ko/ko mice with a C57Bl/6 background were bred from homozygous parents in the animal facility of the Jena University Hospital. To confirm knockout of Terc, DNA was extracted from the tail of newborn mice using the DNeasy Blood & Tissue Kit (Qiagen, Venlo, Netherlands) according to the manufacturers protocol. The DNA was then used for genotyping via PCR using the following primer sequences: mTR-R: 5′-TTC TGA CCA CCA CCA ACT TCA AT-3′; mTR-WT-F: 5′-CTA AGC CGG CAC TCC TTA CAA G-3′; 5PPgK(-F): 5′-GGG GCT GCT AAA GCG CAT-3′. The results were analyzed with an Agilent 2200 TapeStation system using a Agilent D1000 ScreenTape (both Agilent Technologies, Santa Clara, CA, USA). The WT Terc product had a size of 200 bp while the knockout product had a size of 180 bp. Only mice that showed a single band at 180 bp were used for the further breeding process. The Terc ko/ko mice were used for infection at the age of 8 weeks. Young WT (age 8 weeks) and old WT (age 24 months) C57Bl/6 mice were purchased from Janvier Labs (Le Genest-Saint-Isle, France).
All mice were anesthetized with 2% isoflurane before intranasal infection with S. aureus USA300 (1x10 8 CFU/ml) per mouse. After 24 hours, the mice were weighed and scored as previously described (Hornung et al., 2023) . Infected Terc ko/ko mice were grouped into different degrees of severity based on their clinical score, fatal outcome of the disease (fatal) and the presence of bacteria in organs other than the lung (systemic infection). The mice were sacrificed via injection of an overdose of xylazine/ketamine and bleeding of axillary artery. BAL was collected by instillation and subsequent retrieval of PBS into the lungs. Blood and organs were collected. The organs were stored at -80°C until further usage. Mice cohorts were not blinded to those performing the experiments.
Third-generation (G3) female Terc ko/ko mice with a C57Bl/6 background were bred from homozygous parents in the animal facility of the Jena University Hospital. To confirm knockout of Terc, DNA was extracted from the tail of newborn mice using the DNeasy Blood & Tissue Kit (Qiagen, Venlo, Netherlands) according to the manufacturers protocol. The DNA was then used for genotyping via PCR using the following primer sequences: mTR-R: 5′-TTC TGA CCA CCA CCA ACT TCA AT-3′; mTR-WT-F: 5′-CTA AGC CGG CAC TCC TTA CAA G-3′; 5PPgK(-F): 5′-GGG GCT GCT AAA GCG CAT-3′. The results were analyzed with an Agilent 2200 TapeStation system using a Agilent D1000 ScreenTape (both Agilent Technologies, Santa Clara, CA, USA). The WT Terc product had a size of 200 bp while the knockout product had a size of 180 bp. Only mice that showed a single band at 180 bp were used for the further breeding process. The Terc ko/ko mice were used for infection at the age of 8 weeks. Young WT (age 8 weeks) and old WT (age 24 months) C57Bl/6 mice were purchased from Janvier Labs (Le Genest-Saint-Isle, France).
All mice were anesthetized with 2% isoflurane before intranasal infection with S. aureus USA300 (1x10 8 CFU/ml) per mouse. After 24 hours, the mice were weighed and scored as previously described (Hornung et al., 2023) . Infected Terc ko/ko mice were grouped into different degrees of severity based on their clinical score, fatal outcome of the disease (fatal) and the presence of bacteria in organs other than the lung (systemic infection). The mice were sacrificed via injection of an overdose of xylazine/ketamine and bleeding of axillary artery. BAL was collected by instillation and subsequent retrieval of PBS into the lungs. Blood and organs were collected. The organs were stored at -80°C until further usage. Mice cohorts were not blinded to those performing the experiments.
RNA was extracted from murine lung tissue using the Trizol/Chloroform method. First, about 15 mg of lung tissue was homogenized in TriZol LS reagent (Thermo Fisher Scientific, Waltham, MA, US) using the SpeedMill Plus. The tissue-free supernatant of the homogenate was transferred into a new tube after centrifugation. 50 µl of chloroform was added to 250 µl TriZol Reagent, incubated for 3 minutes at RT, and centrifuged at 12000xg for 15 minutes at 4°C. The aqueous phase was mixed with isopropanol in a 1:2 ratio and incubated at RT for 10 minutes before centrifugation at 12000xg for 10 minutes at 4°C. Afterward, the RNA pellet was washed twice with 75% ethanol. Finally, ethanol was removed, and the pellet was air-dried for 5-10 minutes. The dry pellet was then dissolved in sterile distilled water, and RNA concentration was determined by an ND-1000 NanoDrop spectrophotometer (PEQLAB Biotechnologie GmbH, Erlangen, Germany). Before sequencing, RNA integrity was measured using a 5400 Fragment Analyzer (Agilent Technologies, Santa Clara, CA, US)) RNA concentration and RNA integrity of the samples can be found in Supplemental Table 1.
Library construction and mRNA sequencing were performed by Novogene Co., LTD. (Beijing, China) using the Illumina platform Novaseq 6000 S4 flowcell V1.0, based on the sequencing by synthesis (SBS) mechanism and PE150 strategy. For bioinformatics analysis, raw reads in FASTQ format were first processed using fastp. Reads containing adapters and poly N sequences were removed as well as low quality reads to generate clean data. Clean reads were then mapped to the reference genome (Mus Musculus (GRCm39/mm39)) with the HISAT2 software. Gene expression levels were quantified using the FPKM method. DESeq2 software as well as negative binomial distribution was used for analysis of differential gene expression. For FDR (False discovery rate) calculation the Benjamini-Hochberg procedure was used. A summary of the quality of the sequencing data of each sample can be found in Supplemental Table 2.
Heatmaps of the differentially regulated genes (DEG) were constructed using R (v4.2.2; R Foundation for Statistical Computing, https://www.r-project.org/). Only significant DEGs (p-value < 0.05) were displayed in the heatmaps. Non-significant genes were set to zero and are shown in white. All DEGs used for construction of the respective heatmaps are summarized in Supplemental Table 3a-i.
Library construction and mRNA sequencing were performed by Novogene Co., LTD. (Beijing, China) using the Illumina platform Novaseq 6000 S4 flowcell V1.0, based on the sequencing by synthesis (SBS) mechanism and PE150 strategy. For bioinformatics analysis, raw reads in FASTQ format were first processed using fastp. Reads containing adapters and poly N sequences were removed as well as low quality reads to generate clean data. Clean reads were then mapped to the reference genome (Mus Musculus (GRCm39/mm39)) with the HISAT2 software. Gene expression levels were quantified using the FPKM method. DESeq2 software as well as negative binomial distribution was used for analysis of differential gene expression. For FDR (False discovery rate) calculation the Benjamini-Hochberg procedure was used. A summary of the quality of the sequencing data of each sample can be found in Supplemental Table 2.
Heatmaps of the differentially regulated genes (DEG) were constructed using R (v4.2.2; R Foundation for Statistical Computing, https://www.r-project.org/). Only significant DEGs (p-value < 0.05) were displayed in the heatmaps. Non-significant genes were set to zero and are shown in white. All DEGs used for construction of the respective heatmaps are summarized in Supplemental Table 3a-i.
T cells and AMs were isolated as described above. Cells were grown on poly-L-lysine (Merck-Millipore, Burlington, MA, US) coated glass coverslips in a 1:1 ratio. T cells were allowed to attach to the coverslips for 30 minutes before adding S. aureus. After adhesion of T cells, S. aureus USA300 was added at a MOI1 and incubated at 37°C. After 3.5 hours, the samples were fixed with freshly prepared modified Karnovsky fixative (4% w/v paraformaldehyde, 2.5 % v/v glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4) for 1 hour at RT. After washing each 3 times for 15 minutes with 0.1 M sodium cacodylate buffer (pH 7.4), the cells were post-fixed with 2% (w/v) osmium tetroxide for 1 hour at RT. Subsequently, the samples were dehydrated in ascending ethanol concentrations (30, 50, 70, 90, and 100%) for 15 minutes each. Next, the samples were critical-point dried using liquid CO 2 and sputter coated with gold (thickness approx. 2 nm) using a CCU-010 sputter coater (safematic GmbH, Zizers, Switzerland). Finally, the specimens were investigated with a field emission SEM LEO-1530 Gemini (Carl Zeiss NTS GmbH, Oberkochen, Germany).
The length of telomeres in the lung of young WT, old WT and Terc ko/ko mice was measured using the Absolute Mouse Telomere Length Quantification qPCR Assay Kit (ScienCell research Laboratories, Carlsbad, CA, USA) according to the manufacturer's protocol. Genomic DNA from the lungs was extracted by DNeasy Blood & Tissue Kit (Qiagen, Venlo, Netherlands) according to the manufacturer's protocol. For the PCR 2ng of each sample used. Average telomere length per chromosome end was calculated as described by the manufacturer.
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More about "Telomerase RNA component"
The Telomerase RNA Component (TERC) is a non-coding RNA molecule that plays a crucial role in the maintenance of telomeres, the protective caps at the ends of chromosomes.
This essential component of the telomerase enzyme complex serves as a template for the reverse transcription of telomeric DNA sequences, ensuring the preservation of telomeres during cell division.
Understanding the structure, function, and regulation of TERC is key to advancing research in cellular aging, cancer, and stem cell biology.
TERC works in conjunction with the enzyme terminal deoxynucleotidyl transferase (TdT) to add telomeric repeats to the ends of chromosomes, a process known as telomere elongation.
Researchers often utilize techniques like the BD Vacutainer CPT tubes, Magnapure Isolation Kit, and SYBR Green assay to isolate and analyze TERC.
The NanoDrop ND-1000 and NanoDrop 2000c spectrophotometers are commonly used to quantify and assess the purity of TERC samples.
The PrimeScript RT reagent kit can be employed for reverse transcription, and the Hybond-N+ membrane is useful for Northern blot analysis of TERC expression.
In some studies, researchers may leverage GP2-293 packaging cells to produce viral vectors for delivering TERC-related genetic material.
By understanding the key aspects of TERC and the associated experimental techniques, researchers can optimize their investigations into cellular immortality, age-related diseases, and cancer.
This essential component of the telomerase enzyme complex serves as a template for the reverse transcription of telomeric DNA sequences, ensuring the preservation of telomeres during cell division.
Understanding the structure, function, and regulation of TERC is key to advancing research in cellular aging, cancer, and stem cell biology.
TERC works in conjunction with the enzyme terminal deoxynucleotidyl transferase (TdT) to add telomeric repeats to the ends of chromosomes, a process known as telomere elongation.
Researchers often utilize techniques like the BD Vacutainer CPT tubes, Magnapure Isolation Kit, and SYBR Green assay to isolate and analyze TERC.
The NanoDrop ND-1000 and NanoDrop 2000c spectrophotometers are commonly used to quantify and assess the purity of TERC samples.
The PrimeScript RT reagent kit can be employed for reverse transcription, and the Hybond-N+ membrane is useful for Northern blot analysis of TERC expression.
In some studies, researchers may leverage GP2-293 packaging cells to produce viral vectors for delivering TERC-related genetic material.
By understanding the key aspects of TERC and the associated experimental techniques, researchers can optimize their investigations into cellular immortality, age-related diseases, and cancer.