All gingival tissues were collected as described above using a bacterial DNA extraction kit (Tiangen, China) and SYBR Green kit (Cwbio, China) to evaluate the relative content of P. gingivalis in the different groups using RT-PCR. The primers for universal bacteria 16s rRNA and P. gingivalis 16s rRNA are described in Table 1 .
Sybr green kit
Manufactured by CWBIO
Sourced in China
The SYBR Green kit is a laboratory reagent used in real-time PCR (polymerase chain reaction) assays. It is a fluorescent dye that binds to double-stranded DNA, allowing for the quantification of DNA amplification during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Bacterial dna extraction kit, by Tiangen Biotech (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Reverse transcription system, by Vazyme (1 mentions)
Cfx96 detection system, by Bio-Rad (1 mentions)
Trizol reagent, by Tiangen Biotech (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Hifi mmlv cdna kit, by CWBIO (1 mentions)
4 protocols using sybr green kit
1
Quantifying P. gingivalis in Gingival Tissue
2
Quantifying Inflammatory Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from cells using Trizol reagent (Thermo Fisher Scientific, United States) according to the manufacturer’s instructions. RNA (1 μg) was reverse transcribed into cDNA by Reverse Transcription System (Vazyme, Nanjing, China). To quantify the mRNA levels of inflammatory factors, real-time PCR was performed using SYBRGreen Kit (Cwbio, Beijing, China) on a Bio-Rad CFX96 Detection System. The relative gene expression was normalized to β-actin. Data were analyzed by using the comparative Ct method. The primers of target genes used in this study were listed in Supplementary Table 1 .
3
Evaluation of Virulence Factors in Porphyromonas gingivalis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The in vitro experiments were divided into 4 groups based on the OD600 value. Different groups were co-cultured in BHI in the presence of 10% glucose or 1% or 0.1% stevioside. The control group was cultured in BHI, and all bacterial solutions were filled to 2,000 µL with BHI and collected after 36 h. The bacteria were collected in centrifuged tubes and centrifuged at 10,000 rpm for 1 min, following which a bacterial DNA extraction kit (Tiangen, China) and SYBR Green kit (Cwbio, China) were used to evaluate the relative expressions of Fim-A and Hag-A in different groups via RT-PCR. The primers of different genes are all listed in Table 1 .
Primers sequences of different genes in the RT-PCR
| Primers | Sequence (5’ to 3’) |
|---|---|
| Gapdh F | TGCACCACCAACTGTTA |
| Gapdh R | GATGCAGGGATGATGTT |
| TNF-α F | CCACGTCGTAGCAAACCAC |
| TNF-α R | TTGTCCCTTGAAGAGAACCTG |
| IL-1β F | CAGGCAGGCAGTATCACTCA |
| IL-1β R | TGTCCTCATCCTGGAAGGTC |
| IL-6 F | CCGGAGAGGAGACTTCACAG |
| IL-6 R | TCCACGATTTCCCAGAGAAC |
| U-16 S rRNA F | ACTCCTACGGGAGGCAGCAGT |
| U-16 S rRNA R | ATTACCGCGGCTGCTGGC |
| Pg 16 S rRNA F | TGGGTTTAAAGGGTGCGTAG |
| Pg 16 S rRNA R | CAATCGGAGTTCCTCGTGAT |
| HagA-F | ACAGCATCAGCCGATATTCC |
| HagA-R | CGAATTCATTGCCACCTTCT |
| FimA-F | TACTTCCACGCCTTCTCCTGTT |
| FimA-R | CATCTTTACTGTTGCCACTTCG |
4
Quantitative Real-Time PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total RNA was extracted by Trizol reagent (TIANGEN, DP419). Reverse transcription was performed using HiFi-MMLV cDNA Kit (Cwbio, CW0744M) to synthesize the cDNA template. PCR primers used for PCR amplification were obtained from Sangon Biotech (Chengdu, China). Quantitative real-time PCR was performed using SYBR Green Kit (Cwbio, CW2601H) in LightCycler 480 (Roche). The reaction program contained 40 cycles of denaturation at 95°C for 15 s and annealing at 60°C for 1 min. The RNA expression levels were calculated by the 2−ΔΔCt(Quantitation−ComparativeCT) method based on the normalization of GAPDH values. The primer sequences used in our study are as follows: TP53: Forward primer: 5′-AGTGGGAATCTTCTGGGACG-3′, Reverse primer: 5′-TCTTTTGCTGGGGAGAGGAG-3′; GAPDH: Forward primer: 5′-CAAGGCTGAGAATGGGAAGC-3′, Reverse primer: 5′- GAAGACGCCAGTAGACTCCA-3′.
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!