Home Protocols High Specific Activity Radiolabeled RNA Probe Synthesis (E2040)

High Specific Activity Radiolabeled RNA Probe Synthesis (E2040)

Introduction

The HiScribe T7 High Yield RNA Synthesis Kit can be used to synthesize high specific activity radiolabeled RNA probes by following the modified protocol below. More than 50% of the label can be incorporated in a 10 minute reaction. The labeled RNA probes have a specific activity of about 108 cpm/μg.

Protocol

  1. Choosing 32P labeled nucleotide:

    We recommend using [α-32P] UTP or CTP at 800–6000 Ci/mmol and ≥ 10 mCi/ml for the synthesis of radiolabeled RNA probes. We do not recommend using radiolabeled ATP or GTP since less label is generally incorporated. RNA labeled with [α-32P] ATP or GTP appears to be more subject to decomposition during storage.

  2. Thaw the necessary kit components, mix and pulse-spin in microfuge to collect solutions to bottom of tubes. Keep on ice.

  3. Dilute 100 mM UTP to 40 μM UTP if [α-32P] UTP is used.

    1. Prepare 400 μl of 1 mM UTP by combining 4 μl of 100 mM UTP and 396 μl of nuclease-free water. Extra 1 mM UTP solution can be stored at -20°C for future use.

    2. Prepare 100 μl of 40 μM UTP by combining 4 μl of 1 mM UTP and 96 μl of nuclease-free water.

  4. Prepare master mix. For accurate pipetting we recommend preparing 15 μl master mix at a minimum, which is enough for 5 labeling reactions. The master mix contains reaction buffer, ATP, GTP and CTP.

    5. Nuclease-free water 10 μl
    10X Reaction Buffer 2 μl
    ATP (100 mM) 1 μl
    GTP (100 mM) 1 μl
    CTP (100 mM) 1 μl
    Total reaction volume 15 μl
    Use 3 μl master mix for each labeling reaction. Extra master mix can be stored at -20°C for future use.

  5. Assemble the reaction at room temperature in the following order:

    6. Nuclease-free water X μl
    Master Mix 3 μl 1 mM each A, G and C, final
    UTP (40 μM) 2 μl 4 μM final
    [α-32P] UTP X μl 0.2 to 1 μM final
    Template DNA X μl 0.1 to 1μg
    DTT (0.1M)*
    		 1μl
    		 5 mM
    T7 RNA Polymerase Mix 1 μl  
    Total reaction volume 20 μl  

    *Addition of DTT (5mM final) to the reaction is optional but recommended.The RNA polymerase in the kit is sensitive to oxidation and could result in lower RNA yield over time due to repeated handling etc. Adding DTT to the reaction may help restore the kit performance in such cases. Adding DTT will not compromise the reaction in any situation.

    The labeled NTP is present at a limiting concentration and is therefore referred to as the “limiting nucleotide.” (e.g. UTP here). The “limiting nucleotide” is a mixture of both the labeled and unlabeled form of that NTP. There is a trade-off between synthesis of high specific activity probe and synthesis of full-length probe. The higher the concentration of the “limiting nucleotide”, the higher the proportion of full-length transcripts, but if unlabeled nucleotide is used to increase the “limiting nucleotide” concentration, it will lower the specific activity of the transcript. For most labeling reactions, use of 4–5 μM of the “limiting nucleotide” is necessary for full-length probe synthesis with high specific activity. The template sequence will also affect the specific activity of the transcript. For example, if the transcript contains more UTP, more 32P-UTP will be incorporated and the specific activity will be higher.

  6. Table 2. Concentration of [α-32P] NTP in a transcription reaction

    7. Specific Activity
    (Ci/mol)    		 Concentration
    (mCi/ml)    		 Volume used per
    20 μl Reaction    		 Concentration in
    20 μl Reaction
    (hot label)
    800 10 1 μl 0.63 μM
    800 20 1 μl 1.25 μM
    800 40 1 μl 2.5 μM
    3000 10 1 μl 0.17 μM
    3000 20 1 μl 0.33 μM
    3000 40 1 μl 0.67 μM
    6000 40 1 μl 0.33 μM

    Mix thoroughly; pulse-spin in microfuge and incubate for 10 minutes. Incubation temperature is not crucial for labeling efficiency. Room temperature to 40°C can be used.

  7. Optional: To remove template DNA, add 2 μl of DNase I (RNase-free) (NEB #M0303), mix and incubate for 15 minutes at 37°C.

  8. Proceed with purification of synthesized RNA or analysis of transcription products by gel electrophoresis (for purification, we recommend the Monarch RNA Cleanup Kits (NEB #T2040 or #T2050)).