Gene Expression/Protein Analysis

Shanghai Model Organisms provides high-quality gene and protein expression analysis services, including RT-PCR, real time PCR, and Western blot, with accurate experimental results that can be used for publication.

Reverse transcription PCR (RT-PCR) is a technique that combines reverse transcription (RT) of RNA with the polymerase chain reaction (PCR) of cDNA. First, cDNA is synthesized from RNA via the action of reverse transcriptase. Then, cDNA is used as a template to amplify target DNA fragment through multiple steps and multiple cycles, including denaturation, annealing and extension. During the course of this reaction, the yield of target DNA increases exponentially, making it possible to detect some extremely small amounts of RNA.

We provide:

1. Extracted RNA and reverse transcribed cDNA samples

2. Operating procedures, reagents and consumables

3. Primer sequences

4. Agarose electropherogram

5. Raw data and formal experimental data obtained after processing


Service cycle: 7 business days

Note:

1. For cell samples, their size should be more than 106 cells and the samples should be stored at -80 °C after collection. For tissue samples, 100 mg of each sample are required. For serum or blood samples, at least 1 ml of each sample is required and the samples should be transported on dry ice

2. More than 10 samples are required for a single gene

3. The service cycle does not include the time required for the synthesis of probes and standard substances

4. The default extraction method is based on TRIZOL. If the extraction needs to be carried out using another type of assay kit, it should be provided by the customer

5. The specimens can be saved at our company free of charge for one month but will be handled at our discretion after one month. If the samples need to be mailed, the relevant cost should be paid by the customer.


Real-time quantitative PCR is a method in which a fluorescent group is added to the PCR reaction system to monitor the entire PCR process based on the accumulation of fluorescence signals. The amount of an unknown template can be quantitatively analyzed based on a standard curve. Alternative, a relative quantification method can be used based on the ΔΔCt ratio between the target gene and the internal reference gene. The technology not only achieves the quantification of the RNA template, but also has the characteristics of high sensitivity, high specificity, high reliability, capability of multiple reactions, a high degree of automation, non-polluting, real-time and high accuracy.


Experimental steps:

1. Sample collection, processing and preparation

1.1 Suspension cells: Pour the culture medium containing 1×106 cells/ml of suspension cells into a centrifuge tube and precipitate the cells by centrifugation. Rinse twice with PBS, and centrifuge the cells at the bottom of the centrifuge tube. Add 500-1000 μl Trizol into the centrifuge tube and store the sample in a -80 °C freezer. Alternatively, store the stem cells at the bottom of the centrifuge tube in a -80 °C freezer.

1.2 Adherent cells: Use trypsin solution containing 1×106 cells/ml of trypsinization cells, rinse twice with centrifugation PBS, and centrifuge the cells at the bottom of the centrifuge tube. Add 500-1000 μl Trizol into the centrifuge tube and store the sample in a -80 °C freezer. Alternatively, store the stem cells at the bottom of the centrifuge tube in a -80 °C freezer.

1.3 Tissues: Collect 100 mg of fresh tissue and put it into a centrifuge tube. Label the tube and freeze the tissue sample in liquid nitrogen before storing the sample in a -80 °C freezer.

1.4 Blood: Collect 1 ml of whole blood with an anticoagulation tube and add an RNA protective agent. Label the tube and store the sample in a -80 °C freezer.

2. Quality control of RNA or DNA

3. Reverse transcription

4. Primer validation

5. qPCR procedure

6. Data analysis


We provide:

1. Extracted RNA, its electropherogram and reverse transcribed cDNA samples

2. Operating procedures, reagents and consumables

3. Primer sequences

4. Dissolution curve and amplification curve

5. Raw data and formal experimental data obtained after processing


Western blotting is a method used to identify specific antigens by combining the electrophoresis technology used for protein separation with the immunolabeling technology. 


Experimental steps:

1. Protein extraction and quantification from cell or tissue samples.

2. SDS-PAGE

3. Membrane blotting

4. Block the membrane

5. Immune reaction

6. Membrane scan and image acquisition

7. Data analysis


We provide:

1. Remaining samples

2. Experimental report

3. Fluorescence or grayscale image of the original protein bands


Experimental instruments:

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Odyssey infrared fluorescence imaging system


Sample result:

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Service cycle: 1 month


Note:

1. More than 106 cells are required for each cell sample. Stem cells should be collected and stored in a -80 °C freezer or in liquid nitrogen

2. 100 mg or more tissue samples are required for each sample, which should be stored in a -80 °C freezer or in liquid nitrogen

3. Primary antibodies and nuclear (membrane) protein extraction kits should be provided by the customer.

4. The internal reference is provided free of charge (GAPDH is usually used as the internal reference)


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