Total RNA was isolated from human gingival tissues and primary culture human GF using TRIzol reagent (Ambion, Carlsbad, CA, USA). Non-inflamed or inflamed human gingival tissues were homogenized in TRIzol reagent using a glass tissue grinder. RNA was reverse-transcribed, and the complementary DNA was amplified by PCR using Taq polymerase (GeneAll, Songpa, Seoul, Republic of Korea). Quantitative real-time PCR (qRT-PCR) was performed using an iCycler (Bio-Rad Laboratories, Hercules, CA, USA) and the SYBR premix Ex Taq (Takara Bio, Kyoto, Japan). All qRT-PCR was performed in duplicate, and the target gene amplification signal was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the same reaction. The relative levels of SIRT family (SIRT1–7) gene expression were analyzed using the comparative Ct (cycle threshold) method. The average Ct was calculated for SIRT1 to SIRT7 and GAPDH, as previously described.26 (link) Primers and experimental conditions are shown in Supplementary Table 1 . Western blot analysis was performed to detect the cellular and secreted levels of NAMPT, COX-2 and MMP3 in total cell lysates or in conditioned culture media using standard techniques. The following primary antibodies were used for western blotting: NAMPT (AdipoGen), MMP3 (Abcam), COX-2 (Cayman) and TUBULIN (Sigma-Aldrich).
Taq polymerase
Manufactured by GeneAll
Taq polymerase is a thermostable DNA polymerase enzyme isolated from the bacterium Thermus aquaticus. It is a commonly used enzyme in polymerase chain reaction (PCR) techniques for the amplification of DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Power sybr green pcr master mix, by Toyobo (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Icycler, by Bio-Rad (1 mentions)
Cox 2, by Cayman Chemical (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Nampt, by Adipogen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Reverse transcription system, by Promega (1 mentions)
Image quant software program, by GE Healthcare (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rt premix, by iNtRON Biotechnology (1 mentions)
96 automated capillary electrophoresis systems, by Agilent Technologies (1 mentions)
Hq buffer, by GeneAll (1 mentions)
Prosize 2 0 version 1, by Agilent Technologies (1 mentions)
Exgene plant sv mini kit, by GeneAll (1 mentions)
7 protocols using taq polymerase
1
Profiling SIRT Family Expression in Gingival Tissues
2
Silver Material Effects on NALP3 and Caspase-1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PMA-primed THP-1 cells (1×107) were treated with 100 µg/mL of silver materials for 24 h and then, all RNA was extracted from the cells using an RNeasy Mini Kit (Qiagen, Germany) in accordance with the manufacturer's protocol. Extracted RNA was reverse transcribed using the Reverse Transcription System (Promega, USA). Synthesized cDNA was amplified by PCR using Taq polymerase (GeneAll, Korea). The sequence of specific primers for NALP3 (cytoplasmic receptor) and GAPDH was as follows: NALP3 forward: 5′-TGCCTTTGACGAGCACATAG-3′ ; NALP3 reverse: 5′-GCAGCAAACTGG AAGGAAG-3′ ; caspase-1 forward: 5′-GAAGGCATTTGTGGGAAGAA-3′ ; caspase-1 reverse: 5′-CATCTGGCTGCTCAAATGAA-3′ ; and GAPDH forward: 5′-GAGTCAACGGATTTGGTCGT-3′ ; GAPDH reverse: 5′-TTGATTTTGGAGGGATCTCG-3′ . After 5 min at 95°C, 28 cycles at 95°C for 1 min, 53°C for 1 min (caspase-1; 51°C), and 72°C for 2 min were performed, ending with a final extension step at 72°C for 5 min. Quantification of PCR products was performed by electrophoresis. Data was analyzed using the Image Quant software program (GE Healthcare, USA).
3
RT-PCR and RT-qPCR Analysis of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The prepared cDNAs were used for RT-PCR with Taq polymerase (GeneALL, Seoul, Republic of Korea) and gene-specific primers (Table S3 ). After initial denaturation at 95 °C for 5 min, PCR was performed using a thermocycler (Step One Plus Real-Time PCR System; Applied Biosystems) with 35 amplification cycles (95 °C for 1 min, 50–55 °C for 30 s, and 72 °C for 1 min) and finalized with an additional extension step at 72 °C for 10 min. The PCR products were assessed by 1% agarose gel electrophoresis. RT-qPCR was performed as per the procedures noted by Bustin et al. [14 (link)] using Power SYBR Green PCR Master Mix (Toyobo, Osaka, Japan) with gene-specific primers (Table S3 ). In addition, after initial heat treatment at 95 °C for 2 min, qPCR was performed with 40 cycles of denaturation at 95 °C for 30 s, annealing at 50−55 °C for 30 s, and extension at 72 °C for 30 s. The elongation factor 1 (EF1, Table S3 ) was used as a reference gene to normalize the expression level of each qPCR sample. Further, quantitative analyses were performed using the comparative CT (2−ΔΔCT) method [15 (link)]. All experiments were independently replicated three times.
4
Analysis of SePOX gene expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After the absence of DNA contamination was confirmed by PCR with the RNA template, the first strand cDNA was synthesized from the RNA extract (1 µg per reaction) by reverse transcription using RT-premix (Intron Biotechnology, Seoul, Korea) containing an oligo dT primer (5′- CCAGTGAGCAGAGTGCGAGGACTCGAGCTCAAGCTTTTTTTTTTTTTTTTT-3′ ). Ten SePOX genes (SePOX-A∼SePOX-J) were analyzed by RT-PCR using the cDNAs. All PCRs used 40 amplification cycles under 94°C denaturation for 30 sec, gene-specific annealing temperatures for 30 sec and 72°C extension for 30 sec using Taq polymerase (GeneAll, Seoul, Korea) with gene-specific primers (Table 2 ).
5
Microsatellite Analysis of Sheep Genetic Diversity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In the present study, a total of 21 microsatellite loci, 14 of which are
recommended by FAO (2011) to determine genetic diversity in sheep, were
used. PCR amplifications were carried out in a total volume of 25 L
PCR mixture. PCR mixture consisted of 2–2.5 L genomic DNA (50 ng L ), 1.2 L HQ buffer (Geneall), 2 L dNTPs (2.5 mM L ),
0.25 L of each primer (10 pmol L ), 0.4 L (2.5 U L) Taq polymerase
(Geneall), and distilled deionized water. PCR reaction conditions were
performed as follows: initial denaturation at 95 C for 5 min,
followed by 30 cycles of denaturation at 94 C for 45 s,
annealing (at 50–60 C for different loci) for 45 s,
extension at 72 C for 45 s, and final extension at
72 C for 5 min.
In this study, 96 automated capillary electrophoresis systems
(Advanced Analytical Technologies, Iowa, USA) were used for fragment
analysis. The capillary conditioning solution, inlet buffer, separation gel,
and 35–500 bp marker were prepared according to the user manual provided by
the manufacturer. After capillary electrophoresis separation, the raw data
were recorded, and band sizes were calculated using PROSize®2.0 version 1.3.1.1 (Advanced Analytical Technologies, Iowa, USA).
recommended by FAO (2011) to determine genetic diversity in sheep, were
used. PCR amplifications were carried out in a total volume of 25 L
PCR mixture. PCR mixture consisted of 2–2.5 L genomic DNA (50 ng L ), 1.2 L HQ buffer (Geneall), 2 L dNTPs (2.5 mM L ),
0.25 L of each primer (10 pmol L ), 0.4 L (2.5 U L) Taq polymerase
(Geneall), and distilled deionized water. PCR reaction conditions were
performed as follows: initial denaturation at 95 C for 5 min,
followed by 30 cycles of denaturation at 94 C for 45 s,
annealing (at 50–60 C for different loci) for 45 s,
extension at 72 C for 45 s, and final extension at
72 C for 5 min.
In this study, 96 automated capillary electrophoresis systems
(Advanced Analytical Technologies, Iowa, USA) were used for fragment
analysis. The capillary conditioning solution, inlet buffer, separation gel,
and 35–500 bp marker were prepared according to the user manual provided by
the manufacturer. After capillary electrophoresis separation, the raw data
were recorded, and band sizes were calculated using PROSize®2.0 version 1.3.1.1 (Advanced Analytical Technologies, Iowa, USA).
6
RT-PCR and RT-qPCR Protocol for Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For RT-PCR, the cDNAs were amplified in specific target genes using Taq polymerase (GeneAll Biotechnology Co., Ltd., Seoul, Republic of Korea) with gene-specific primers (Supplementary Table S2 ). The PCR reaction was initiated with an initial denaturation at 95°C for 5 min. The subsequent 35 amplification cycles were performed at 95°C for 1 min, 53–55°C for 1 min, and 72°C for 1 min. The PCR reaction was finalized with an additional extension step at 72°C for 10 min. The PCR product was analyzed by 1% agarose gel electrophoresis to determine the presence of the amplified product. For RT-qPCR, the Power SYBR Green PCR Master Mix (Toyobo, Osaka, Japan) was used with gene-specific primers under the guidelines of Bustin et al. (2009) (link). Quantitative analysis was performed using the comparative CT method (Livak and Schmittgen, 2001 (link)). All experiments were independently replicated three times.
7
Hibiscus DNA Isolation and Amplification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Leaves of the five different Hibiscus cultivars were placed in liquid nitrogen for 1 min and crushed into powder. Genomic DNA was isolated using the Exgene ™ Plant SV mini kit (GeneAll Biotechnology Co., Seoul, Korea), according to the manufacturer's instructions. The primer pairs used for the amplification of trnL-F region were synthesized by Macrogen (Seoul, Korea); the sequences of the universal primers are listed in Table 2 [16] (link). The PCR reaction mixture consisted of 50 ng of DNA, 0.5 µM of primers, 10 µL of Taq polymerase (GeneAll Biotechnology Co., Seoul, Korea), and 5 µL of distilled water. PCR products were analyzed by electrophoretic separation in a submarine horizontal agarose slab gel apparatus. For running the DNA samples, a gel strength of 2% agarose was dissolved in 0.5 X TAE buffer followed by heating in microwave oven. After cooling to approximately 55 °C, 0.05 µL/mL GelGreen ® Nucleic Acid Gel Stain (Fremont, CA, USA) was added. Then, 300 mL of 0.5X TAE buffer was poured to sufficiently submerge the gel in the electrophoresis tank. Electrophoresis was performed at a constant voltage of 5 V/cm until the bromophenol dye reached almost the end of the gel. A UV-trans illuminator was used to visualize the DNA fragments of different sizes separated in the gel (Fig. 1).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!