Human GAPDH qPCR Primer Pair，即人GAPDH qPCR引物对，主要用于基于SYBR Green的qPCR、One-Step qRT-PCR或semi-quantitative PCR。本引物为预先设计、经过qPCR验证、预混的引物对。
qPCR (Quantitative PCR)即定量PCR，也称实时荧光定量PCR或实时定量PCR (Real-time quantitative PCR)、实时PCR (Real-time PCR)，是一种在DNA扩增反应过程中，以荧光定量测定每个聚合酶链式反应(PCR)循环后产物总量的方法。qPCR常用的两种方法是SYBR Green等荧光染料法和探针法。SYBR Green等荧光染料法是使用带有荧光的、非特异的DNA结合染料SYBR Green等以检测PCR过程中积累的PCR扩增产物；而探针法(Probe method)，也常被称为TaqMan探针法，不使用荧光染料，而采用荧光基团和淬灭基团(Quencher)标记的DNA探针靶向拟通过PCR检测的目标序列。
对于SYBR Green等染料法，引物至关重要。本系列引物产品采用阿拉丁开发的引物设计算法，优化了序列并经过验证，特异性佳，扩增效率高，引物二聚体形成发生率低，qPCR数据可靠。本系列引物大多数扩增产物(Amplicon)的长度约100-150 bp。本产品为预混冻干粉，每管含正向引物(Forward primer，也称上游引物)和反向引物(Reverse primer，也称下游引物)各1 nmol，共2 nmol，不含核酸酶(Nuclease-free)，只需加入400 μL超纯水溶解成2.5 μM each，即可使用。按20 μL或25 μL体系使用2 μL引物，本产品每管可以用于200次qPCR实验。
Gene Information
Gene Name | glyceraldehyde-3-phosphate dehydrogenase |
Gene Symbol | GAPDH |
Synonyms | G3PD; GAPD; HEL-S-162eP |
Organism | Human |
Gene ID | 2597 |
UniProt ID | P04406 |
Main Accession No. | NM_002046 |
Other Accession No. | NM_001256799, NM_001289745, NM_001289746, NM_002046, NM_001357943, NR_152150, NM_002046.1, NM_002046.2, NM_002046.3, NM_002046.4, NM_002046.5, NM_001256799.1, NM_001256799.2, NM_001289745.1, NM_001289746.1, BC009081, BC009081.1, BC001601, BC004109, BC013310, BC020308, BC023632, BC025925, BC026907, BC029340, BC029618, BC083511, BE893087, BG724119, BI463134, BM763361, BT006893, BU155402, NM_001289745.3, NM_002046.7, NM_001289746.2, NM_001256799.3 |
Map Location | 12p13 |
Pathway | Housekeeping gene, used for internal control |
Gene Summary | This gene encodes a member of the glyceraldehyde-3-phosphate dehydrogenase protein family. The encoded protein has been identified as a moonlighting protein based on its ability to perform mechanistically distinct functions. The product of this gene catalyzes an important energy-yielding step in carbohydrate metabolism, the reversible oxidative phosphorylation of glyceraldehyde-3-phosphate in the presence of inorganic phosphate and nicotinamide adenine dinucleotide (NAD). The encoded protein has additionally been identified to have uracil DNA glycosylase activity in the nucleus. Also, this protein contains a peptide that has antimicrobial activity against E. coli, P. aeruginosa, and C. albicans. Studies of a similar protein in mouse have assigned a variety of additional functions including nitrosylation of nuclear proteins, the regulation of mRNA stability, and acting as a transferrin receptor on the cell surface of macrophage. Many pseudogenes similar to this locus are present in the human genome. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Nov 2014] |
Amplicon Information
Amplicon Length (bp) | 130 |
NCBI mRNA ID | NM_002046.7 |
NCBI Protein ID | NP_002037.2 |
Ensembl Transcript ID | ENST00000229239.10 |
Ensembl Gene ID | ENSG00000111640.15 |
Ensembl mRNA ID | GAPDH-201 |
注意事项：1. PCR扩增产物的长度可能会因基因转录后存在多种剪接形式而有所差异。
2. 虽然本系列引物产品的特异性好，但仍建议进行熔解曲线(Melt curve)分析以确定扩增反应的特异性。如果只有一个熔解曲线峰(对应的退火温度即双链DNA产物的Tm值)，说明只有一种单一产物；如果熔解曲线出现双峰、多峰或杂峰峰，可能是引物二聚体或非特异性扩增、存在基因组DNA污染、试剂及环境被污染等。建议设置不含模板的对照(No template control, NTC)，即反应体系中包含除模板以外的所有反应组分，根据样品孔和无模板对照孔熔解曲线的差异，可判断是否存在引物二聚体或其它的非特异性扩增。
3. 若反应体系存在扩增产物污染，推荐使用 防污染型 UltraBio™ SYBR Green qPCR Mix(S751589) 。
4. 本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。
5. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
使用说明：
1. PCR反应体系的设置：
(1) 开启本产品前，3000-8000×g离心1分钟，以防开盖时引物干粉散失。每管加入400 μL超纯水，先盖好盖子颠倒混匀数次，然后离心机快速离心几秒，开盖后再轻轻吹打混匀，即得400 μL 2.5 μM each的Primer Mix。
(2) 融解并混匀PCR反应所需的各种溶液。SYBR Green qPCR Mix需完全融解并混匀后置于冰浴上或冰盒内。推荐使用 UltraBio™ SYBR Green qPCR Mix (S751589)。
(3) 参考下表在室温或冰浴上设置PCR反应体系，以96孔板和UltraBio™ SYBR Green qPCR Mix为例。
Reagent | Volume for One PCR Reaction |
UltraBio™ SYBR Green qPCR Mix(2X) | 10μl |
Primer Mix (2.5μM each) | 2μl |
Template DNA | 2μl |
RNase-Free Water | 6μl |
Total Volume | 20μl |
注：
1. 通常引物的终浓度为0.2-0.5 μM each时可获得良好的检测效果，也可根据情况在0.1-1.0 μM each范围内调整引物的终浓度。
2. 通常DNA模板的量以1-10ng cDNA为参考用量。因不同物种的模板中含有的目的基因拷贝数不同，如有必要，可加大模板用量或对模板进行梯度稀释，以确定最佳的模板使用量。RT-PCR反应得到的cDNA直接作为模板时，其添加量不要超过PCR反应总体积的10%。
3.96孔板的推荐反应体系为20 µL，也可以根据实际实验需求，按比例扩大或缩小反应体系。
4. 建议设置不加模板的阴性对照组。
(4) 用移液器轻轻吹打混匀或轻微Vortex混匀，室温离心数秒，使液体积聚于管底。
(5) 将设置好的PCR反应管置于荧光定量PCR仪上，开始定量PCR反应。
2. PCR反应程序：
在Real-time PCR反应前进行模板的预变性，通常设定为95ºC 2分钟，复杂或高GC模板适当延长时间至5-10分钟。
(1) 预变性：95ºC 2分钟；
(2) 变性：95ºC 15秒；
(3) 退火/延伸：60ºC 15-30秒；
(4) 重复步骤(2)和步骤(3) ，总共40个循环；
(5) 熔解曲线分析(可选)：95ºC 15秒, 60ºC 15秒, 95ºC 15秒；
(6) 使用荧光定量PCR仪提供的软件分析结果。
注：以上举例为常规qPCR反应系统，仅供参考。实际反应条件因模板、引物等的结构不同而各异，需根据模板、引物、目的片段的特点设定最佳反应条件，并根据比例放大或缩小反应体系。上述为两步法qPCR，如果采用三步法qPCR，只需在退火/延伸后加一步72ºC 30秒，随后重复步骤 (2)、(3)及增加的这一步骤共40个循环即可。
Human GAPDH qPCR Primer Pair is primarily designed for SYBR Green‑based qPCR, one‑step qRT‑PCR, or semi‑quantitative PCR. This primer pair is pre‑designed, qPCR‑validated, and supplied as a premixed primer pair.
qPCR (Quantitative PCR), also known as real‑time fluorescent quantitative PCR or real‑time PCR, is a method that quantifies the total product accumulated after each polymerase chain reaction (PCR) cycle during DNA amplification using fluorescence detection. The two common approaches for qPCR are fluorescent dye‑based methods (e.g., SYBR Green) and probe‑based methods. SYBR Green‑based methods use a non‑specific fluorescent DNA‑binding dye (such as SYBR Green) to detect the accumulating PCR amplicons during the reaction. The probe method (often called the TaqMan probe method) does not use fluorescent dyes but instead employs DNA probes labelled with a fluorophore and a quencher that target the specific sequence to be detected by PCR.
For dye‑based methods like SYBR Green, the primers are critical. This series of primer products is developed using Aladdin’s proprietary primer design algorithm, with optimised sequences that have been validated to ensure good specificity, high amplification efficiency, low incidence of primer‑dimer formation, and reliable qPCR data. Most amplicons in this series are approximately 100–150 bp in length. This product is supplied as a pre‑mixed lyophilised powder. Each tube contains 1 nmol of forward primer and 1 nmol of reverse primer (total 2 nmol), is nuclease‑free, and can be reconstituted by adding 400 μL of ultrapure water to obtain a 2.5 μM each solution ready for use. When using 2 μL of primer mix per 20 μL or 25 μL reaction, each tube provides sufficient material for 200 qPCR reactions.
Gene Information
Gene Name | glyceraldehyde-3-phosphate dehydrogenase | Gene Symbol | GAPDH | Synonyms | G3PD; GAPD; HEL-S-162eP | Organism | Human | Gene ID | 2597 | UniProt ID | P04406 | Main Accession No. | NM_002046 | Other Accession No. | NM_001256799, NM_001289745, NM_001289746, NM_002046, NM_001357943, NR_152150, NM_002046.1, NM_002046.2, NM_002046.3, NM_002046.4, NM_002046.5, NM_001256799.1, NM_001256799.2, NM_001289745.1, NM_001289746.1, BC009081, BC009081.1, BC001601, BC004109, BC013310, BC020308, BC023632, BC025925, BC026907, BC029340, BC029618, BC083511, BE893087, BG724119, BI463134, BM763361, BT006893, BU155402, NM_001289745.3, NM_002046.7, NM_001289746.2, NM_001256799.3 | Map Location | 12p13 | Pathway | Housekeeping gene, used for internal control | Gene Summary | This gene encodes a member of the glyceraldehyde-3-phosphate dehydrogenase protein family. The encoded protein has been identified as a moonlighting protein based on its ability to perform mechanistically distinct functions. The product of this gene catalyzes an important energy-yielding step in carbohydrate metabolism, the reversible oxidative phosphorylation of glyceraldehyde-3-phosphate in the presence of inorganic phosphate and nicotinamide adenine dinucleotide (NAD). The encoded protein has additionally been identified to have uracil DNA glycosylase activity in the nucleus. Also, this protein contains a peptide that has antimicrobial activity against E. coli, P. aeruginosa, and C. albicans. Studies of a similar protein in mouse have assigned a variety of additional functions including nitrosylation of nuclear proteins, the regulation of mRNA stability, and acting as a transferrin receptor on the cell surface of macrophage. Many pseudogenes similar to this locus are present in the human genome. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Nov 2014] |
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Amplicon Information
Amplicon Length (bp) | 130 | NCBI mRNA ID | NM_002046.7 | NCBI Protein ID | NP_002037.2 | Ensembl Transcript ID | ENST00000229239.10 | Ensembl Gene ID | ENSG00000111640.15 | Ensembl mRNA ID | GAPDH-201 |
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Precautions
1.The length of the PCR amplicon may vary due to the existence of multiple splicing isoforms after gene transcription.
2.Although this series of primer products has good specificity, we still recommend performing a melt curve analysis to confirm the specificity of the amplification reaction. If only a single melt curve peak (corresponding to the melting temperature, T<sub>m</sub>, of the double‑stranded DNA product) is observed, it indicates a single specific product. If the melt curve shows double peaks, multiple peaks, or irregular peaks, it may indicate primer dimers, non‑specific amplification, genomic DNA contamination, or contamination of reagents or the environment. We recommend including a no‑template control (NTC) – i.e., a reaction containing all components except the template – so that by comparing the melt curves of sample wells and the NTC well, the presence of primer dimers or other non‑specific amplification can be judged.
3.If amplicon contamination is present in the reaction system, we recommend using the contamination‑proof UltraBio™ SYBR Green qPCR Mix (S751589).
4.This product is for professional scientific research use only. It must not be used for clinical diagnosis or treatment, nor for food or drug applications, and should not be stored in ordinary domestic premises.
5.For your safety and health, please wear a lab coat and disposable gloves when handling.
Instructions for Use
1. Setting up the PCR reaction system
(1) Before opening the product, centrifuge the tube at 3,000–8,000 × g for 1 minute to prevent loss of primer powder upon opening. Add 400 μL of ultrapure water to each tube, close the cap and invert to mix several times, then briefly centrifuge. Open the cap and gently pipette to mix thoroughly. This yields 400 μL of Primer Mix at 2.5 μM each.
(2) Thaw and mix all solutions required for the PCR reaction. The SYBR Green qPCR Mix must be completely thawed and mixed before placing on ice or in an ice bath. We recommend using UltraBio™ SYBR Green qPCR Mix (S751589).
(3) Prepare the PCR reaction system at room temperature or on ice according to the table below. The example uses a 96‑well plate and UltraBio™ SYBR Green qPCR Mix.
Reagent | Volume for One PCR Reaction | UltraBio™ SYBR Green qPCR Mix(2X) | 10μl | Primer Mix (2.5μM each) | 2μl | Template DNA | 2μl | RNase-Free Water | 6μl | Total Volume | 20μl |
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Notes:
1.Typically, a final primer concentration of 0.2–0.5 μM each gives good detection results; the concentration can be adjusted within the range of 0.1–1.0 μM each as needed.
2.The recommended amount of DNA template is generally 1–10 ng of cDNA. Because the copy number of the target gene varies among different species, you may increase the template amount or perform serial dilutions to determine the optimal template quantity. When using cDNA directly from an RT‑PCR reaction, its volume should not exceed 10% of the total PCR reaction volume.
3.The recommended reaction volume for 96‑well plates is 20 μL; you may scale the reaction up or down proportionally according to actual experimental needs.
4.It is recommended to include a no‑template negative control.
(4) Gently pipette or vortex briefly to mix, then centrifuge at room temperature for a few seconds to collect the liquid at the bottom of the tube.
(5) Place the prepared PCR reaction tubes in a real‑time PCR instrument and start the quantitative PCR reaction.
2. PCR reaction program
Prior to the real‑time PCR reaction, pre‑denature the template – typically set at 95 °C for 2 minutes; for complex or high‑GC templates, extend the time appropriately to 5–10 minutes.
(1) Pre‑denaturation: 95 °C for 2 minutes
(2) Denaturation: 95 °C for 15 seconds
(3) Annealing/Extension: 60 °C for 15–30 seconds
(4) Repeat steps (2) and (3) for a total of 40 cycles
(5) Melt curve analysis (optional): 95 °C for 15 seconds, 60 °C for 15 seconds, 95 °C for 15 seconds
(6) Analyse the results using the software provided with the real‑time PCR instrument.
Note: The above example is a conventional qPCR reaction system and is for reference only. Actual reaction conditions vary depending on the structure of the template, primers, etc. Optimal conditions should be determined according to the specific features of the template, primers, and target fragment, and the reaction system can be scaled up or down proportionally. The above is a two‑step qPCR; if a three‑step qPCR is preferred, simply add a 72 °C for 30 seconds step after the annealing/extension step, then repeat steps (2), (3) and this additional step for a total of 40 cycles.
ZJ26F0737206
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