you will have to retrotranscribe the RNA, amplify it by PCR, then clone the PCR product in a plasmid downstream a T7 promoter (you can add it on the direct primer you will used for amplification of the RT product )... lot of work; could be easier to get the DNA directly (from addgen for example) If the DNA fragment is not too long it can be direclty synthesized (and cloned) by a private company ?

es, you can generate a quantified RNA stock for real-time RT-PCR using your DNA positive control, but using ssRNA directly for this purpose is not ideal. Here’s a step-by-step guide to achieve your goal:

Steps to Generate a Quantified RNA Stock from a DNA Positive Control:

Step 1: Amplify the Target DNA (If Needed)

• If your DNA positive control contains the full target sequence, you can proceed.
• If it’s a plasmid or contains extra sequences, use PCR to amplify only the target region.
• Purify the PCR product using a PCR purification kit.

Step 2: Perform In Vitro Transcription (IVT) to Generate RNA

• Use an in vitro transcription kit (like T7, SP6, or T3 RNA polymerase-based kits) if your DNA template has the corresponding promoter.
• Transcribe the DNA into RNA following the kit instructions.
• Treat the RNA with DNase I to remove any residual DNA.
• Purify the RNA using an RNA purification kit.

Step 3: Quantify and Validate the RNA

• Measure RNA concentration using a Nanodrop, Qubit, or a spectrophotometer.
• Check RNA integrity using agarose gel electrophoresis or a Bioanalyzer.

Step 4: Create Aliquots and Store the RNA

• Divide the RNA into small aliquots to prevent degradation from multiple freeze-thaw cycles.
• Store at -80°C for long-term stability.

Can You Use ssRNA Instead?

• If you have an ssRNA and want to convert it back to RNA, you would need to:Reverse transcribe it into cDNA using reverse transcriptase. Amplify the cDNA using PCR. Perform in vitro transcription to generate new RNA.
• However, this process is unnecessary if you already have a DNA positive control, which is more stable and directly usable for RNA transcription.

Recommendations

✅ Use your DNA positive control for in vitro transcription—it’s the most reliable method. ❌ Avoid using ssRNA as a starting material unless absolutely necessary. ✅ Ensure proper quantification and storage to maintain RNA integrity.

Let me know if you need more details on any step! 😊

Achieving your desired outcome might not be entirely straightforward. Using DNA as a control could be a good option, as it's generally easier to amplify than RNA. However, if you specifically need an RNA control for reverse transcription, you could consider using RNA extracted from a cell line already available in your lab. This would usually be simpler than reverse transcribing your own RNA and then performing in vitro transcription (which, incidentally, would require adding a promoter sequence at the 5' end). Alternatively, if you don't need to assess a large number of genes, using a plasmid for your standard curve could be another viable approach.

**Single-stranded RNA (ssRNA)** can be converted into **complementary DNA (cDNA)** through **reverse transcription**, and then the cDNA can be used as a template to synthesize **RNA** again. This process is widely used in molecular biology, virology, and vaccine development (e.g., mRNA vaccines like Pfizer/Moderna).

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### **Step-by-Step Process: ssRNA → cDNA → RNA**

#### **1. Converting ssRNA to cDNA (Reverse Transcription)**

- **Enzyme Used:** Reverse Transcriptase (RT, e.g., M-MLV RT, HIV RT).

- **Key Requirements:**

- A **primer** (oligo-dT for poly-A tails or gene-specific primers).

- Deoxynucleotide triphosphates (**dNTPs**).

- RNA template (intact, high-quality ssRNA).

- **Process:**

- The primer binds to the ssRNA.

- Reverse transcriptase synthesizes a complementary DNA strand (cDNA).

- Result: A **RNA-cDNA hybrid** (which can be degraded by RNase H or kept intact).

#### **2. Converting cDNA Back to RNA (In Vitro Transcription, IVT)**

- **Enzyme Used:** DNA-dependent RNA polymerase (e.g., **T7, T3, or SP6 RNA polymerase**).

- **Key Requirements:**

- A DNA template (cDNA) with a **promoter sequence** (e.g., T7 promoter).

- Ribonucleotide triphosphates (**NTPs**: ATP, UTP, GTP, CTP).

- Buffer with Mg²⁺ for polymerase activity.

- **Process:**

- The RNA polymerase binds to the promoter and synthesizes RNA from the cDNA template.

- Result: **Newly synthesized RNA** (identical or complementary to the original ssRNA, depending on the template).

---**Key Applications**

✔ **Vaccine Production** (e.g., mRNA vaccines – synthetic mRNA is made from cDNA templates).

✔ **Virus Research** (e.g., studying RNA viruses like SARS-CoV-2, HIV, or influenza).

✔ **Gene Expression Studies** (RT-qPCR, RNA sequencing).

✔ **Synthetic Biology** (designing artificial RNA molecules).

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**Important Notes**

⚠ **RNA Quality Matters**: Degraded ssRNA leads to incomplete cDNA synthesis.

⚠ **Enzyme Choice**: Some reverse transcriptases degrade RNA (RNase H activity), while others preserve it (RNase H– mutants).

⚠ **Template Design**: For efficient RNA synthesis, the cDNA must have a strong promoter (e.g., T7).