1 Introduction

The CIS/SOCS family of intracellular proteins are represented by eight members (CIS and SOCS1-7) (1, 2) and are involved in the negative regulation of Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signalling pathways. They are expressed in response to multiple cytokines and growth factors, and have an important role in regulating many biological systems, including hemopoietic development and the innate and adaptive immune response, particularly in the context of bacterial and viral infections. Thus far, CIS, SOCS1, SOCS2, and SOCS3 are the best-characterized family members. STAT binding to specific elements in the promoter regions of SOCS genes results in a rapid increase in both mRNA and protein, with the SOCS proteins often acting to inhibit the initiating cytokine as part of a classic negative-feedback loop (1, 3). CIS, SOCS1, SOCS2, and SOCS3 inhibit signalling by binding to phosphorylated tyrosine residues within the receptor cytoplasmic domains to block further STAT recruitment (CIS/SOCS2) (4, 5), or alternatively bind directly to activated JAK kinases, inhibiting catalytic activity (SOCS1/3) (6–8). In addition, the SOCS proteins act as adaptors to bring the SOCS box-associated E3 ubiquitin ligase complex in contact with an SH2-bound substrate, targeting bound proteins for ubiquitination and degradation via the proteasome (9–11).

The expression level of SOCS proteins varies in different cells and tissues, with for instance, SOCS1 protein only detectable in the thymus under normal (non-stimulatory) conditions. If mice are injected with cytokines such as granulocyte colony stimulating factor (G-CSF), interferon (IFN) γ or interleukin (IL)-6, then expression of SOCS mRNA is easily detected in various tissues (1, 3). In BMDM, SOCS2, SOCS4, and SOCS5 are expressed at a low to moderate level, whereas CIS, SOCS1, and SOCS3 are undetectable in the absence of stimulation. Treatment of BMDM with various stimuli results in the rapid upregulation of SOCS mRNA, often within short timeframes (0.5–4 h). SOCS1 is induced in response to IFNγ, SOCS3 in response to IL-6 family members and Toll-like receptor (TLR) ligands such as lipopolysaccharide, whilst SOCS2 and CIS are upregulated in response to IL-4 (3, 12, 13). Regulation of the SOCS genes provides a biological context to understand their function and is of a great interest in different aspects of biomedical research including cancer, inflammation, autoimmunity and response to infection (14–16).

Following the discovery of PCR (1985) (17, 18) and then qPCR (1993) (19), the latest technique has evolved and at present combines PCR with many fluorescent detection systems to quantify the mRNA level (absolute or relative). In a basic PCR the amplified product is detected in the end of the cycling program, whereas in RT-qPCR, the DNA can be detected after each cycle and quantified by measuring the associated fluorescent signal. The intensity of fluorescence increases in proportion with the number of cycles. The results can be presented as an Amplification Plot where the Ct value defines the cycle number at which the amplified product reaches the threshold. The threshold is the point at which sample fluorescence can be detected above background fluorescence. The Ct value is related to the initial amount of DNA and inversely proportional to the expression level of the gene. If the Ct value is low, it means the fluorescence reaches the threshold early, and the amount of target in the sample is high. The Ct is therefore dependent on the threshold level and it is important to maintain a ­consistent threshold value for the same gene from one run to another (20).

In this chapter we describe RT-qPCR analysis of SOCS genes using the SYBR® Green I method; SOCS1 expression in IL-4-stimulated BMDM is shown as an example. The following main aspects are outlined:

  1. 1.

    The principles of successful primer and amplicon design.

  2. 2.

    RNA purification and cDNA synthesis.

  3. 3.

    Preparation of the standard curve and sample analysis.

  4. 4.

    Standard curve quantification and normalization against a reference gene (RG).

2 Materials

Prepare all solutions using nuclease-free water. RNA purification, cDNA synthesis, and PCR setup can be performed at room temperature (unless indicated). Fine pipettes P2, P10 and a multichannel P10 should be used to reduce variation between samples. Use filter tips only.

Reagents and equipment:

  1. 1.

    70% Ethanol (EtOH).

  2. 2.

    14.3 M ß-mercaptoethanol (ß-ME).

  3. 3.

    RNA purification kit, includes RLT buffer and columns (see Note 1).

  4. 4.

    SYBR® Green I master mix (see Note 2).

  5. 5.

    Forward and Reverse primers (5 μM stock solutions).

  6. 6.

    Reverse transcriptase (RT) (50 U/μl) and buffer (see Note 3).

  7. 7.

    RNase-Free DNase (30 U/μl).

  8. 8.

    Ribonuclease inhibitor (RNasin) (40 U/μl).

  9. 9.

    dNTP mix (dATP, dGTP, dCTP, and dTTP) (10 mM each).

  10. 10.

    Oligo (dT)14–20 (50 μM).

  11. 11.

    Reaction Mix I: 1 μl of Oligo (dT)14-20 (50 μM) and 1 μl of dNTP mix.

  12. 12.

    Reaction Mix II: 4 μl 5× RT buffer, 2 μl of 0.1M DTT, 1 μl of RNAsin, and 1 μl of RT.

  13. 13.

    Standard amplicons at 0.01 pmol/4 μl (0.0025 μM in water).

  14. 14.

    Eight dilutions of the Standard amplicons from 10−2 pmol/4 μl to 10−9 pmol/4 μl.

  15. 15.

    0.1 M Dithiothreitol (DTT).

  16. 16.

    Sterile 1.5 mL Eppendorf tubes.

  17. 17.

    Microcentrifuge (for 1.5 and 2 ml tubes) and macrocentrifuge (suitable for 384- or 96-well plates).

  18. 18.

    Optical adhesive film (transparent tape) for sealing the plates.

  19. 19.

    Plate stand.

  20. 20.

    Heating blocks set up with the temperature 50  ºC, 65  ºC, and 80  ºC.

  21. 21.

    384- and 96-well plates.

  22. 22.

    PCR thermocycler and corresponding software (see Note 4).

  23. 23.

    Disposable gloves.

3 Methods

3.1 Principles of Successful Primer and Amplicon Design

As SYBR® Green I binds to dsDNA and the method is highly sensitive, it is important to design primers which are specific for the gene of interest. The following general principles can be applied to any primer/amplicon design:

  1. 1.

    To avoid amplification of contaminating genomic DNA, primers should, where possible, be designed to cross intron–exon boundaries. If at least one primer can anneal across the intron–exon boundary, then only cDNA will be amplified (see Note 5). Alternatively, the forward primer can be designed to hybridize to one exon and the reverse primer to the second exon. The amplicon from cDNA will be smaller than that from genomic DNA and amplified much more efficiently. The greater the difference in size of the amplification product between cDNA and genomic DNA, the more accurate the results are likely to be.

  2. 2.

    Primer length should be between 17–25 bp.

  3. 3.

    GC content should be 50–60%.

  4. 4.

    The primer annealing temperature (Tm) should be 50–60  ºC.

  5. 5.

    Avoid repeats and mismatches as well as complimentary sequence stretches within and between primers.

  6. 6.

    Avoid sequences that will result in the formation of hairpins or primer duplexes.

  7. 7.

    Verify the uniqueness of the primer sequence by blasting it against the NCBI nucleotide database (www.ncbi.nlm.nih.gov/BLAST/).

  8. 8.

    The amplicon should be less than 200 bp (80–150 bp is optimal). There is a balance between having a short amplicon with higher PCR efficiency, and a longer one which will incorporate more SYBR Green I and give greater detection sensitivity.

    Table 1 SOCS primers
    Full size table
  9. 9.

    The GC content of the amplicon should be 40–60%.

  10. 10.

    Avoid amplicons with predicted secondary structure. Secondary structure can be analyzed using the following program:

    (http://molbiol-tools.ca/Repeats_secondary_structure_Tm.htm).

  11. 11.

    Sequences of forward and reverse primers for the SOCS genes are provided in Table 1.

3.2 Total RNA Purification and cDNA Synthesis (Two Step RT-qPCR)

RNA quality, as assessed by purity and integrity, has a significant impact on RT-qPCR performance (12). Genomic DNA contamination can be an issue with any RNA purification method, as amplification from a trace amount of DNA can lead to misinterpretation of the results, especially if there is variation in the amount of genomic DNA between the samples (see Subheading 3.1 and Note 1). We use a two-step qPCR where the mRNA sample is reverse transcribed in a separate tube to the PCR reaction. This allows the expression of multiple genes to be analyzed from one reverse transcribed sample.

  1. 1.

    Purify RNA using the RNAeasy or RNAeasy Plus kits, according to the manufacturer’s instructions, essentially as follows. Alternate methods for RNA purification can also be used (see Note 1).

  2. 2.

    For mouse cells, we recommend starting with 1  ×  106 cells/sample; however, it can be adjusted depending on the cell type. Lyse cells in 350 μl of RLT lysis buffer containing 143 mM of ß-mercaptoethanol (1:100 dilution from stock) for 5 min at room temperature (see Note 6). Harvest lysates into Eppendorf tubes, add 300 μl of 70% EtOH, mix by pipetting and load onto the column. Alternatively, lysates can be stored in RLT buffer at −80 °C for at least 6 months.

  3. 3.

    Elute RNA from the column in 10 μl of nuclease-free water (depending on the expected yield/Ct value, the volume can be increased up to 50 μl) (see Note 7).

  4. 4.

    Determine the concentration of RNA by measuring the absorbance at 260 nm (A260) in a spectrophotometer. Take 1–5 μg of total RNA for cDNA synthesis.

  5. 5.

    Prepare Reaction Mix I, multiplied by the number of samples (see Note 8), mix by vortexing and spin briefly, 30 s at ≥8,000  ×  g.

  6. 6.

    Prepare Reaction Mix II (see Note 9), multiplied by the number of samples, mix by vortexing and centrifuge for 30 s at ≥8,000  ×  g.

  7. 7.

    Add 2 μl of Reaction Mix I to 10 μl of total RNA, mix by pipetting and incubate at 65 °C for 5 min (see Note 11).

  8. 8.

    Transfer the samples to ice (+4  ºC), cool down for 5 min and spin briefly at ≥8,000  ×  g.

  9. 9.

    Add 8 μl of Reaction Mix II, pipette, spin briefly and incubate at 50  ºC for 40 min to 1 h.

  10. 10.

    Terminate the reaction by heating the samples at 80  ºC for 5 min, chill on ice, and centrifuge briefly at ≥8,000  ×  g.

  11. 11.

    Dilute the cDNA if necessary by adding 80 μl of nuclease-free water (see Note 10).

3.3 Preparation of the Standard Curve and Sample Assessment

There are several ways to obtain the amplicon for the standard curve: (a) A DNA fragment based on the cDNA sequence amplified by your primers can be ordered from a commercial company; (b) The cDNA can be amplified from a tissue or cells in which your gene of interest is highly expressed; (c) You can use a recombinant DNA plasmid, which contains the cloned gene of interest. For (b) and (c), the DNA fragment should be gel purified and its concentration determined by measuring the absorbance at 260 nm (A260). The sequences of standard amplicons for the SOCS genes are provided in Table 2.

Table 2 Standard amplicons
Full size table
Table 3 Example data analysis
Full size table
  1. 1.

    Prepare a dilution series of each standard amplicon. We recommend a tenfold dilution range (see Note 12). Add 450 μl of nuclease-free water to seven Eppendorf tubes. Transfer 50 μl from the 0.0025 μM amplicon solution to the second tube to obtain a 0.00025 μM solution. Mix by pipetting/vortexing, spin briefly, change the tip and transfer 50 μl into another tube to obtain 0.001 pmol/4 μl etc.

  2. 2.

    For each gene, prepare a Master Mix by adding 0.5 μl each of Forward and Reverse primers to 5 μl of SYBR® Green I Master Mix. Multiply by the number of samples. Using a multichannel pipette, transfer 6 μl of the Master mix as required, to each well of a 384-well plate.

  3. 3.

    Add 4 μl of Standard amplicon or cDNA template (unknown sample) to each well on the plate (generally we run our standard and samples in technical triplicate and duplicates, respectively; with three biological replicates for each sample) (see Note 13).

  4. 4.

    Seal the plate with transparent tape (optical adhesive film).

  5. 5.

    Spin the plate for 30 s at >8,000  ´  g at room temperature.

  6. 6.

    Run on a PCR thermocycler: (95°C for 10 min, 94°C for 15 s, Tm for 25 s, 72°C for 10 s) for 40 cycles.

  7. 7.

    Analyze data (Subheading 3.4).

3.4 Standard Curve Quantification and Normalization to a Reference Gene

The SYBR® Green I system coupled with a STD curve using a known amount of amplicon enables relative quantification (amount/cell number or total RNA). Once the PCR reaction is complete, the Ct value is converted to an amount of cDNA using the standard curve (for an example see Fig. 1 and Table 3). In order to correct for variation between samples, the amount of cDNA should be normalized to a housekeeping or reference gene (RG), the expression of which does not alter under the studied conditions (21–23). There is an extensive list of genes that have been used as RGs (21), but in each case the chosen gene should be tested for regulation under your experimental conditions.

Fig. 1.
figure 00171

Example standard curves (a) and amplification (b) plots for GAPDH and SOCS1. Plots were generated by tenfold serial dilution of the corresponding standard amplicons followed by analysis on an ABI 7900 thermocycler. The slope of the standard curve gives the efficiency of the PCR reaction (Efficiency  =  10(−1/slope) −1). If the slope is −3.32 than PCR efficiency is 100% (24). In this instance the slope is −4.0 for both GAPDH and SOCS1. ΔRn is a measure of the fluorescence associated with SYBR green.

Full size image

We prefer glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for the normalization of SOCS gene expression in BMDM. The amount of expressed GAPDH is easily detectable and unchanged with stimulation (Table 3 and Fig. 2.) (23). We have successfully used porphobilinogen deaminase (PBGD) in other systems, but the level of PBGD expression in unstimulated BMDM is very low (10−8 pmol). Here we use IL-4-induction of SOCS1 (13), to provide an example of the raw data and subsequent analysis generated by RT-qPCR (Table 3).

Fig. 2.
figure 00172

Upregulation of SOCS1 in IL-4-stimulated macrophages. 1.5  ×  106 BMDM (n  =  3) were stimulated with 10 ng/ml IL-4 for 0, 2, 4, 6, 8, and 24 h. Cells were lysed, RNA purified and reverse transcribed, and GAPDH and SOCS1 expression measured by RT-qPCR. Standard curve (a) and Amplification (b) plots for GAPDH and SOCS1 showing positioning of the IL-4-stimulated sample series. In (a), the circles highlight one group of GAPDH samples and two distinct groups of SOCS1 samples (upper group with the higher Ct value reflects SOCS1 expression in untreated cells, whilst the group with the lower Ct value reflects expression in IL-4-stimulated BMDM). In (b) the horizontal line indicates the threshold. (c) The relative expression level of SOCS1 in IL-4-stimulated macrophages. The average amount of SOCS1 has been normalized to the average amount of GAPDH and is expressed as mean  ±  S.D.

Full size image
  1. 1.

    Convert the Ct value to an actual amount using the standard curve.

  2. 2.

    Calculate the average amount obtained from the values for each technical replicate (duplicates/triplicate).

  3. 3.

    Normalize the average amount of the SOCS gene to the average amount of GAPDH for each sample to obtain the relative expression (see Note 14).

  4. 4.

    Take the average relative expression of the biological replicates (the second column from right, Table 2).

  5. 5.

    Calculate the Standard Deviation (STD) for each group of samples.

  6. 6.

    The relative expression level can be represented as in Fig. 2.

4 Notes

  1. 1.

    If primers have been designed based on the intron–exon boundaries, we recommend using the RNAeasy kit from QIAGEN (Valencia, CA). In the case of intronless genes such as SOCS1, SOCS3, or SOCS5, we would recommend using the RNAeasy Plus kit from QIAGEN. In the RNAeasy Plus system, which is designed for mammalian cells, RNA-free DNase is bound to the membrane in the shredding column, significantly decreasing contamination of genomic DNA in the RNA samples. Alternatively, DNAase treatment of the samples can be carried out.

  2. 2.

    We recommend using FastStart Universal SYBR Green Master mix (Rox) (Roche, Mannheim, Germany) due to high sensitivity, reproducibility and reliability (there is minimal variation between batches).

  3. 3.

    We recommend the SuperScript® III First-Strand Synthesis System (Invitrogen, CA) for first strand DNA synthesis from poly A(+) RNA due to the longer life-time and increased thermostability of reverse transcriptase (up to 55 °C). As a result, it enables high cDNA yield and full-length transcripts to be obtained from as little as 1 pg of starting material.

  4. 4.

    We use the ABI Prism 7900 PCR thermocycler and SDS.2 software to analyze our samples.

  5. 5.

    Considering that up to 20% of human genes are either single exon genes or have one or more pseudogenes (basically intronless) (12), it can be problematic to rely only on intron spanning primer design. If your gene of interest undergoes alternative splicing, this should also be taken into consideration when designing primers.

  6. 6.

    As qPCR is a very sensitive technique, it is important to have consistent amounts of RNA in each sample within a dataset. To obtain reproducible results, the same number of cells/volume of samples/organ weight should be taken. RLT is a lysis buffer containing guanidine thiocyanate; it is part of the RNAeasy Plus Mini kit or can be purchased separately from the same company. Add ß-mercaptoethanol to the lysis buffer before use. Dispense it in a fume hood wearing gloves.

  7. 7.

    We recommend converting RNA into cDNA immediately after purification to avoid RNA degradation upon freezing–thawing. Alternatively, RNA can be stored at −80°C.

  8. 8.

    To take into account variations in pipetting, prepare up to 10% excess volume for all master mixes.

  9. 9.

    We recommend including at least one extra sample without RT to determine the level (if any) of genomic DNA in the sample. A RG can be used as a positive control to confirm sample integrity.

  10. 10.

    The optimal dilution factor can vary and we recommend that it be optimized according to your source of RNA and Q-PCR settings.

  11. 11.

    The total PCR reaction volume is 10 μl, and it is therefore desirable to pipette no less than 2 μl to avoid significant variation.

  12. 12.

    All of the SOCS amplicons (Table 2) can be ordered from commercial sources. The standard curve should cover the complete range of expected expression. If the expression level is unknown, we recommend initially using a wide-ranging STD curve. Depending on the expression level of your gene of interest, and to increase accuracy, a twofold dilution can also be used. If the annealing temperature of the primer pairs for ­different amplicons is the same or similar (±2  ºC), multiple standard curves can be run simultaneously.

  13. 13.

    Change the tip each time when collecting 4 μl, even from the same sample.

  14. 14.

    For simplicity, we divide one value by the other (SOCS÷RG) to obtain a relative amount. This allows for the expression of multiple genes to be directly compared in each sample and across experiments, as they are being normalized to the same housekeeping gene. However, if absolute quantification (copy number) is required, this needs to be approached slightly differently, see Ginzinger et al. (24), for more details.